PH sensitive electrode for forming wearable PH sensor for wound state diagnosis and application of PH sensitive electrode

Abstract

The invention provides a pH sensitive electrode for forming a wearable pH sensor for wound state diagnosis and application of the pH sensitive electrode. According to the electrode, the problem that a conventional Ir-based electrode has the defect of biological incompatibility is solved, the biocompatibility of an electrode sensitive material is realized by introducing a biocompatible coating material to the surface of an Ir-based material, and the potential use risk of an Ir-based pH sensor in a wearable application scene is solved.

Description

Technical Field

[0001] The present invention belongs to the fields of electrochemical detection and analysis and chemical sensor technology, and relates to a pH-sensitive electrode for forming a wearable pH sensor for diagnosing wound conditions and its application. Background Art

[0002] The treatment and management of wounds, especially chronic wounds, is an important task in the medical field. Currently, the treatment of chronic wounds mainly relies on three-dimensional dressings for long-term care, and the monitoring of the physiological state of the wound surface during the nursing period is a technical problem that is difficult to solve ideally at present. The popular practice in clinical medicine is to continuously observe the wound surface through the experience of medical staff to judge the physiological or pathological state of the wound surface. This method can only make a prognostic judgment on the wound surface state, cannot warn of the impending risk of deterioration, has insufficient accuracy, and must often physically interfere with the wound surface, bringing great pain to the patient. pH is an important physiological and biochemical index of the physiological state of the wound surface. The normal pH of the wound surface is approximately in the range of 4.5 - 6.5. At an appropriate low pH level, it can not only help resist wound infection and inflammation, but also significantly shorten the wound healing cycle; when the natural pH level of the wound surface shows an upward trend, it can indicate that bacterial proliferation has begun on the wound surface; once the wound is at a high pH level, obvious bacterial infection has occurred on the wound surface, and the wound has or is about to be in a poor state such as infection and inflammation. Therefore, timely and accurate measurement of the pH of the wound surface can accurately judge the physiological state of the wound and can also predict whether it is at risk of infection.

[0003] The precise, stable, and rapid quantification of pH is the core task in pH detection. Currently, there are already mature pH sensor products, but due to the need to rely on frequent calibration and the difficulty in achieving planar design, they cannot meet the requirements of the wound state monitoring scenario with relatively high precision and stability. In addition, most pH sensor sensitive materials have low biocompatibility, especially the problem of being unsafe for direct application on the wound surface. These problems have seriously hindered the development of wearable pH sensors for wound care. Summary of the Invention

[0004] To overcome the problem in traditional electrochemical pH sensing technology that it cannot achieve long-term in-situ full-automatic monitoring due to relying on repeated calibration, the purpose of the present invention is to provide a pH-sensitive electrode for forming a wearable pH sensor for diagnosing wound conditions.

[0005] Another purpose of the present invention is to provide an application of the above pH-sensitive electrode in an electrochemical pH sensing method without calibration.

[0006] The sensor provided by the present invention has a planar and flexible design and can be well attached to the wound surface for use; at the same time, the present invention uses a polymer-Ir-IrO x nanoparticle composite to overcome Ir-IrOx its own biocompatibility and improve its electrochemical signal stability, and use Ir-IrO x 's electrochemical activity to indicate the pH of the wound surface. The provided method can overcome the inherent tissue incompatibility of sensitive materials and solve the safety problem of the sensor when used in situ on the wound surface.

[0007] A pH-sensitive electrode for a wearable pH sensor for diagnosing wound status is prepared by the following method:

[0008] (1) Use triethanolamine DMSO solution to adjust the pH of IrCl4 solution to the level of 2.0 - 6.0, add DMSO solution of 3-methyl-1,2-cyclopentanedione, mix evenly and let stand in the dark;

[0009] (2) Heat the mixed solution obtained in step (1) to 75 - 100 °C, add sodium L-ascorbate solution thereto under stirring conditions, react for 0.2 - 4.5 h, collect the precipitate, namely IrNP, and disperse it in water to obtain an IrNP dispersion;

[0010] (3) Prepare an aqueous solution of 4,4'-diamino-5,5''-(dithiophen-2-yl)-2,2'-bipyridine, add this solution to the IrNP dispersion under stirring conditions, react at 45 - 70 °C for 12 - 24 h, after the reaction ends, collect the precipitate, redisperse the precipitate with water, and prepare a dispersion system with a mass fraction of 0.05 - 0.5%;

[0011] (4) Add an aqueous solution containing Ru 3+ to the dispersion system obtained in step (3), immerse the working surface of the clean substrate electrode into this solution, take it out and wash it after thermostatic treatment at 40 - 60 °C for 45 - 80 min, then immerse it into an aqueous solution containing Ru 3+ , and then immerse it into a bipyridine solution for 60 - 180 min to obtain a pH-sensitive electrode denoted as IR.

[0012] Furthermore, the concentration of the IrCl4 solution in step (1) is 5 - 15 mM, more preferably 10 mM; the IrCl4 solution is obtained by dissolving IrCl4 solid in HCl solution.

[0013] Furthermore, the concentration of triethanol in step (1) is 0.1 - 1 M; the concentration of the DMSO solution of 3-methyl-1,2-cyclopentanedione is 0.1 - 0.25 M.

[0014] Furthermore, the volume ratio of the IrCl4 solution to the DMSO solution of 3-methyl-1,2-cyclopentanedione in step (1) is 100:20 - 75.

[0015] Further, the temperature for standing in step (1) is 20 - 35°C, and the standing time is 12 - 36 h.

[0016] Further, the volume ratio of the mixed solution obtained in step (1) to the sodium L - ascorbate solution is 12 - 18:5 - 25;

[0017] Further, the mass fraction of the IrNP dispersion in step (2) is 0.01% - 2%, preferably 0.1 - 1%;

[0018] Further, the reaction time in step (2) is 0.5 - 2.5 h.

[0019] Further, the concentration of the aqueous solution of 4,4'-diamino - 5,5''-(dithiophen - 2 - yl)-2,2'-bipyridine in step (3) is 10 - 20 mM, preferably 15 mM; and its volume ratio to the IrNP dispersion is 2:4 - 12;

[0020] Further, the reaction temperature in step (3) is 50 - 60°C, more preferably 55°C, and the reaction time is 14 - 20 h, more preferably 16 h.

[0021] Further, the mass fraction of the dispersion system in step (3) is 0.1 - 0.5%.

[0022] Further, in the first time of step (4), the concentration of Ru 3+ in the aqueous solution containing Ru 3+ is 1 - 20 mM, preferably 5 - 15 mM, more preferably 10 mM.

[0023] Further, the temperature for the first constant - temperature treatment in step (4) is 45 - 55°C, preferably 50°C, and the time is 50 - 70 min, preferably 60 min.

[0024] Further, in the second time of step (4), the concentration of the aqueous solution of Ru 3+ is 1 - 20 mM, preferably 5 - 15 mM, preferably 10 mM; and the second immersion time is 10 - 20 min.

[0025] Further, the concentration of the bipyridine solution in step (4) is 2 - 10 mM, preferably 6 mM; and the bipyridine solution is prepared with a PBS buffer solution with a pH of 6 - 8.

[0026] Further, the time for immersing in the bipyridine solution in step (4) is 100 - 140 min, preferably 120 min.

[0027] Further, in step (4), the Ru 3+The aqueous solution is obtained by dissolving RuCl3·6H2O in water.

[0028] Furthermore, the substrate electrode in step (4) is at least one of gold, silver, copper, nickel, iridium, platinum, chromium and their alloys, graphite, carbon black, carbon nanotubes, graphene and its derivatives, and indium tin oxide.

[0029] Furthermore, after preparing IrNP in step (2), a modification step is also included. Specifically, IrNP is dispersed in water containing NaOH, D- / L-cysteine and glycerol, and refluxed at 65-100 °C for 16-48 h to obtain IrNP-pS;

[0030] The concentrations of the NaOH, D- / L-cysteine and glycerol in water are 0.15-1.0 M: 2-60 mM: 0.02-0.2 M, preferably 0.15-1.0 M: 5-30 mM: 0.02-0.2 M.

[0031] The mass fraction of the IrNP in water is 0.05-1%, preferably 0.1-0.5%, and more preferably 0.2%.

[0032] By replacing the IrNP dispersion liquid in step (2) with an IrNP-pS dispersion liquid, the electrode IpR can be prepared.

[0033] Furthermore, a protective layer can be added to the surface of IR or IpR. The protective layer material is any one or more of Nafion resin, hyaluronic acid, chitosan, agarose, cellulose, alginic acid, chondroitin sulfate, polyuronide, gelatin, collagen, polyurethane and its derivatives, silk fibroin, polyvinyl alcohol and its derivatives or copolymers. It can enhance the stability of the sensitive material on the electrode surface; improve the hydrophilicity and hydrophobicity of the electrode interface and the anti-interference ability to specific ions; improve the biocompatibility of the electrode surface; and reduce the surface free energy of the electrode.

[0034] An electrochemical pH sensing method without calibration includes the following steps:

[0035] Using an electrode system containing the above pH sensitive electrode and pH insensitive electrode to detect the pH of a pH standard buffer solution;

[0036] The pH indication parameter a.u. is obtained by voltammetry and defined as where E f is the peak potential, HWHM is the half-peak half-width potential, ΔI1 and ΔI2 are the peak current intensities corresponding to the pH sensitive electrode and pH insensitive electrode respectively, and a is the electrode constant;

[0037] Generating a regression curve according to the relationship between the a.u. value and the pH value of the standard buffer solution;

[0038] An electrode system composed of the same pH-sensitive electrode and pH-insensitive electrode is used to detect the wound exudate at the wound surface to be measured, and the pH value is measured according to the regression curve;

[0039] Both the current and potential signals generated by the pH-sensitive electrode will respond to the change of the pH to be measured;

[0040] The current and potential signals generated by the pH-insensitive electrode do not respond to the change of pH.

[0041] Furthermore, both the current and potential signals generated by the pH-sensitive electrode will respond to the change of the pH to be measured and environmental interference factors; the current and potential signals generated by the pH-insensitive electrode do not respond to the change of pH but only respond to the change of environmental interference factors.

[0042] Furthermore, the interference factors mainly include ion species, ion concentration, solution viscosity, strong acids and bases, extreme temperatures, fluorides, sulfides, attachments, biological contaminants, etc. These are all environmental factors that seriously interfere with the measurement accuracy of traditional methods. This solution has good anti-interference performance for these known interference factors.

[0043] The pH-insensitive electrode is at least one of gold, silver, copper, nickel, iridium, platinum, chromium and their alloys, graphite, carbon black, carbon nanotubes, graphene and its derivatives, or indium tin oxide, or is prepared by modifying a functional material or in-situ introducing a functional material on the above electrode materials.

[0044] Furthermore, the functional materials of the pH-insensitive electrode include but are not limited to: at least one of pure metals, alloys, carbon materials, metal chelates and their derivatives, benzene substances and their derivatives, heterocyclic aromatic hydrocarbons and their derivatives, and electrochemically active biomolecules and their derivatives. Further, the functional materials of the pH-insensitive electrode include but are not limited to at least one of silver, gold, gold amalgam, tin-lead alloy, boron-doped diamond, carbon black, glassy carbon, ruthenium bipyridine, iron phenanthroline, (poly)benzenethiol, and poly(4-vinylpyridine).

[0045] Furthermore, the modification method of the functional material on the substrate electrode is at least one of chemical vapor deposition, vacuum sputtering, evaporation plating, electroplating, electrochemical deposition, chemical in-situ synthesis, electrochemical in-situ synthesis, and film formation.

[0046] Furthermore, the electrode system is one of a two-electrode, three-electrode, and four-electrode system.

[0047] Furthermore, the electrode form is a screen-printed electrode, a planar sputtered thin film electrode, a planar MEMS microelectrode, etc.

[0048] Further, the electrode substrate material is at least one of polyester, polycarbonate, polyurethane, polyimide, polyethylene, polyvinylidene fluoride, polytetrafluoroethylene, polyvinyl chloride, paper, silk, nylon, polyethersulfone, polydimethylsiloxane, or ethylene-vinyl acetate copolymer.

[0049] Further, the voltammetry analysis method includes, but is not limited to, at least one of linear voltammetry, cyclic voltammetry, pulse voltammetry, differential pulse voltammetry, and square wave pulse voltammetry.

[0050] Further, for linear voltammetry analysis and cyclic voltammetry, the scanning potential window is -2.5 to +2.5 V; the scanning rate is 0.01 mV / s - 10 V / s; for pulse voltammetry, the potential increment is 0.01 - 1000 mV / s, the potential amplitude is 1 - 500 mV, and the frequency is 1 - 100000 Hz.

[0051] Further, after obtaining data through voltammetry analysis, primary processing is performed on the data, and the processing methods include, but are not limited to, linear filtering and linear parameter extraction; furthermore, the linear filtering includes at least one of differential filtering, high-pass filtering, and low-pass filtering; the linear parameter extraction includes at least one of derivative function analysis and judgment, non-linear fitting judgment, machine learning fitting judgment, and interpolation method judgment, etc.

[0052] Compared with the prior art, the present invention has the following technical solution features:

[0053] (1) The present invention overcomes the problem of the biocompatibility defect of conventional Ir-based electrodes. By introducing a biocompatible coating material on the surface of the Ir-based material, the biocompatibility of the electrode sensitive material is achieved, and the potential risk of using an Ir-based pH sensor in a wearable application scenario is solved.

[0054] (2) The solution provided by the present invention adopts a non-traditional new principle to achieve precise quantification of pH without calibration. The finished product can be used directly out of the factory without any calibration or calibration treatment during the use period, thus fully meeting the in-situ long-term online sensing requirements of the pH of the wound surface. At the same time, the electrode realizes miniaturization and planarization design, which is suitable for use in contact with the wound surface. Description of the Drawings

[0055] Figure 1 Are the characterization results of the Ir-based sensitive material, where (a) is the TEM photo of IrNP in Example 1; (b) is the TEM photo of IrNP-pS in Example 2; (c) is the XPS C1s fine spectrum of IpR in Example 1; (d) is the XPS Ir4f fine spectrum of IpR in Example 1.

[0056] Figure 2Square wave pulse voltammogram (a) of the IpR screen-printed electrode in Britton-Robinson buffer with pH 2 - 12 in Example 4 and the a.u.-pH regression curve (b) plotted after converting the results to a.u. values.

[0057] Figure 3 Process photos (a) and pH change situation (b) of the long-term wearable simulation test with the IpR flexible electrode in contact with the wound surface in Example 5.

[0058] Figure 4 Results of quantitative analysis (a) of the wound health status and synchronous empirical judgment (b) of the IpR screen-printed electrode in Comparative Example 1.

[0059] Figure 5 Change situation of two cytokines during the experimental period in Comparative Example 2, where the left figure is IL-13 and the right figure is IL-4. Detailed implementation mode

[0060] Example 1

[0061] (1) Preparation of IpR screen-printed electrode

[0062] Dissolve IrCl₄ in 0.5M HCl to prepare 100 mL of an aqueous solution containing 10 mM IrCl₄. Adjust the pH of this solution to 4.0 with a DMSO solution containing 0.5M triethanolamine. Add 20 mL of a DMSO solution of 0.2M 3-methyl-1,2-cyclopentanedione. After mixing the mixture evenly, let it stand in the dark at room temperature for 24 h for use. Heat the mixture to 85 °C and gradually add 100 mL of 0.1M sodium L-ascorbate solution dropwise under stirring. After the reaction ends in 2 h, cool the reaction solution to room temperature and then centrifuge it at 12000 rpm for 15 min to collect the sediment to obtain IrNP. Prepare an aqueous solution containing 15 mM 4,4'-diamino-5,5''-(dithiophen-2-yl)-2,2'-bipyridine with 0.1M PBS buffer at pH 7.4. Gradually add 20 mL of this solution dropwise to 80 mL of the IrNP dispersion (mass fraction 0.5%) under stirring and react at 55 °C for 16 h. After the reaction ends, cool the reaction solution to room temperature and centrifuge it at 10000 rpm for 30 min to collect the sediment.

[0063] Redisperse the sediment with pure water to prepare a dispersion system with a mass fraction of 0.25%. Prepare a RuCl₃·6H₂O solution pre-dissolved with 0.1M HCl solution to prepare a solution containing 10 mM Ru 3+The solution. Immerse the clean working electrode of the stone grinding rod into this solution, take it out and wash it after thermostatic treatment at 50 °C for 60 min. Prepare a 10 mM RuCl3·6H2O solution with 0.1 M HCl solution, immerse the working surface of the electrode into the solution, take it out and wash it after maintaining at room temperature for 15 min. Partially immerse the working surface of the electrode into a 6 mM bipyridine solution prepared with 0.1 M PBS buffer at pH 7.0, take it out and wash it after treatment at room temperature for 120 min to make electrode IR. After measurement, it is determined that the electrode constant a of this electrode is 0.176.

[0064] Example 2

[0065] Disperse the IrNP obtained in Example 1 in ultrapure water containing 0.5 M NaOH, 10 mM L-cysteine, and 0.1 M glycerol (the mass fraction of IrNP is 0.2%), place it under reflux treatment at 100 °C for 16 - 48 h, then cool it to room temperature, and centrifuge it at 12000 rpm for 15 min to collect the sediment to obtain IrNP-pS.

[0066] Example 3

[0067] Prepare an aqueous solution containing 15 mM 4,4'-diamino-5,5''-(dithiophen-2-yl)-2,2'-bipyridine with 0.1 M PBS buffer at pH 7.4. While stirring, slowly add 20 mL of this solution dropwise to 80 mL of the aqueous dispersion of IrNP-pS prepared in Example 2 (mass fraction 0.5%), and react at 55 °C for 16 h. After the reaction is completed, cool the reaction solution to room temperature, centrifuge it at 10000 rpm for 30 min, and collect the sediment.

[0068] Prepare a 10 mM RuCl3·6H2O solution with 0.1 M HCl solution, immerse the clean working electrode of the stone grinding rod into this solution, take it out and wash it after thermostatic treatment at 50 °C for 60 min. Prepare a 10 mM RuCl3·6H2O solution with 0.1 M HCl solution, immerse the working surface of the electrode into the solution, take it out and wash it after maintaining at room temperature for 15 min. Partially immerse the working surface of the electrode into a 6 mM bipyridine solution prepared with 0.1 M PBS buffer at pH 7.0, take it out and wash it after treatment at room temperature for 120 min to make electrode IpR. After measurement, it is determined that the electrode constant a of this electrode is 0.178.

[0069] Example 4

[0070] The electrode IpR prepared in Example 3 was used as the working electrode (2×2 mm), the graphite rod counter electrode (2×2 mm), and the Ag reference electrode (2×1 mm) to form a C-C-Ag three-electrode screen-printed electrode system. A Britton-Robinson standard buffer solution with a concentration of 40 mM was prepared, and the pH of the buffer solution was adjusted to 2 - 12 with 0.5 M standard hydrochloric acid and potassium hydroxide. Differential pulse voltammetry was used to measure (the scanning potential was from 800 to -500 mV, the pulse potential increment was 5 mV, the pulse amplitude was 50 mV, and the pulse frequency was 20 Hz.) the changes in current and potential with pH of the polymeric voltammetric pH electrode in the buffer solution sample, and its pH response curve was recorded. The differential pulse voltammogram in the Britton-Robinson standard buffer solution containing 1 wt% bovine serum albumin (BSA) at pH 2 - 12 is shown as Figure 2 (a). The single-chip microcomputer software automatically calculates E f , HWHM, ΔI1, ΔI2, a, and a.u. values according to the curve result data, and records the relationship between the a.u. value and the pH value of the standard buffer solution to generate a regression curve. The results are shown as Figure 2 (b).

[0071] (2) Wound exudate test of the IpR screen-printed electrode

[0072] Collect 10 samples of chronic wound patients aspirated by a vacuum-assisted closure (VSD) treatment device, 5 of which are from patients diagnosed with healthy wounds and the other 5 are from patients diagnosed with wound infections. The IpR screen-printed electrode and a commercially available imported brand precision glass bulb pH meter were used to independently perform quantitative analysis on the sample pH, and the results are listed in Table 1:

[0073] Table 1 Comparison of quantitative detection results of wound exudate pH (N = 5)

[0074]

[0075] It can be seen from the comparison test results that the IpR screen-printed electrode used to measure the pH value of the wound exudate of patients has a very high degree of coincidence with the commercially available imported brand precision glass bulb pH meter, and can accurately reflect the pH level of the wound exudate and the pH conditions of wounds from different sources and different states. In addition, the analysis results found that the auxiliary treatment with physiological saline will slightly reduce the pH of the wound exudate; common bacterial infections will cause a significant increase in the pH of the wound exudate. This observation result is consistent with medical practice and basic research experience, indicating that the IpR screen-printed electrode can accurately reflect the pH situation of the wound exudate.

[0076] Example 5

[0077] (1) Preparation of the IpR flexible electrode

[0078] Dissolve IrCl4 in 0.5 M HCl to prepare 100 mL of an aqueous solution containing 10 mM IrCl4. Adjust the pH of this solution to 4.0 with a DMSO solution containing 0.5 M triethanolamine. Add 20 mL of a 0.2 M solution of 3-methyl-1,2-cyclopentanedione. After the mixture is thoroughly mixed, let it stand in the dark at room temperature for 24 h for later use. Heat the mixture to 95 °C and gradually add 100 mL of a 0.06 M sodium L-ascorbate solution dropwise under stirring. After the reaction is completed, cool the reaction solution to room temperature and then centrifuge it at 10,000 rpm for 20 min to collect the sediment to obtain IrNP. Disperse IrNP in ultrapure water containing 1 M triethanolamine and 15 mM L-cysteine (the mass fraction of IrNP is 0.2%). Place it under reflux at 100 °C for 6 h and then cool to room temperature. Centrifuge it at 10,000 rpm for 30 min to collect the sediment to obtain IrNP-pS. Prepare an aqueous solution containing 15 mM 4,4'-diamino-5,5''-(dithiophen-2-yl)-2,2'-bipyridine with 0.1 M PBS buffer at pH 7.4. Gradually add 20 mL of this solution dropwise to 80 mL of the IrNP dispersion (mass fraction is 0.5%) and react at 55 °C for 16 h. After the reaction is completed, cool to room temperature and centrifuge it at 10,000 rpm for 30 min to collect the sediment.

[0079] Prepare a 10 mM RuCl3·6H2O solution with 0.1 M HCl solution. Partially immerse the working electrode of the clean graphite rod into this solution and keep it at a constant temperature of 80 °C for 10 min, then take it out and wash it. Prepare a 10 mM RuCl3·6H2O solution with 0.1 M HCl solution. Immerse the working surface of the electrode into the solution and keep it at room temperature for 15 min, then take it out and wash it. Partially immerse the working surface of the electrode into a 6 mM bipyridine solution prepared with 0.1 M PBS buffer at pH 7.0 and treat it at room temperature for 120 min, then take it out and wash it to make electrode IR. Using a similar method, replace the IrNP dispersion with IrNP-pS to prepare electrode IpR. After measurement, determine that the electrode constant a of this electrode is 0.181.

[0080] The prepared electrode IpR was used as the working electrode (2×2 mm), a graphite rod as the counter electrode (2×2 mm), and an Ag reference electrode (2×1 mm) to form a C-C-Ag three-electrode screen-printed electrode system. A Britton-Robinson standard buffer solution with a concentration of 40 mM was prepared, and the pH of the buffer solution was adjusted to 2 - 12 with 0.5 M standard hydrochloric acid and potassium hydroxide. Differential pulse voltammetry (scan potential from 800 to -500 mV, pulse potential increment of 5 mV, pulse amplitude of 50 mV, pulse frequency of 20 Hz) was used to test the change of current and potential with pH of the polymeric voltammetric pH electrode in the buffer solution sample, and its pH response curve was recorded. The single-chip microcomputer software automatically calculated E f , HWHM, ΔI1, ΔI2, a, and a.u. values. The relationship between the a.u. value and the pH value of the standard buffer solution was recorded to generate a regression curve, and it was measured that a.u. = -16.02·pH + 5.521, R 2 = 0.998, and this regression equation was used for subsequent tests.

[0081] (2) Contact-type wound surface pH sensing simulation test of the IpR flexible electrode

[0082] Ballistic gelatin was configured to simulate the open wound surface: Gelatin particles were completely dissolved in deionized water to obtain a 30% weight ratio gelatin solution, which was then poured into a self-made mold and naturally cooled to room temperature to form a 30 mm cube. Subsequently, the gelatin cube was transferred to a self-made electrochemical cell ( Figure 3 a). The newly prepared IpR flexible electrode sheet was closely attached and placed above the gelatin and sealed with a lid. Then, the cell was connected to a peristaltic pump to form a circulating, closed system, where artificial wound fluid (artificial wound fluid (AWF) was configured according to the following composition: 2% human serum albumin, 0.36% sodium chloride, 0.05% sodium bicarbonate, 0.02% sodium citrate, 0.1% sodium lactate, 0.1% glucose, 0.01% calcium chloride dihydrate, 0.02% magnesium chloride, 0.01% urea.) was used as the fluid phase. The system was exposed to ultraviolet light overnight for sterilization. The system was continuously tested for 60 days in the SWV mode, and the pH value of the simulated environment was converted through the results. In the experimental group, except that starting from the 20th day, AWF containing 0.04% NaHCO3 was pumped in every day to simulate the wound infection process, and from the 40th to 60th day, AWF containing 0.25% L-lactic acid was pumped in to simulate the wound healing process, the continuous pH change of a parallel system without any treatment was also tested for 60 days.

[0083] The results recorded in the experiment are shown in Figure 3b: In the experimental group, the pH changes recorded by the IpR flexible electrode were consistent with the two characteristic changes in wound surface pH, and the sensor could accurately record the pH value of the wound surface. In the control group, there was no obvious change in the pH recorded by the sensor, and it always remained at the initial value level. This indicates that the sensor can maintain high stability and signal reproducibility in a long-term in-situ test environment and can be used for continuous and accurate measurement of wound surface pH.

[0084] Comparative Example 1

[0085] IpR screen-printed electrode for improving the pre-diagnosis efficiency of chronic wounds in VSD-assisted treatment

[0086] Prepare the electrode IpR screen-printed electrode using the sensor preparation method described in Example 1. Install the electrode inside the supporting liquid storage bottle of the VSD special three-dimensional dressing, and place the end of the electrode outside the bottle and connect it to the sensing device. Select 16 healthy rats with a body weight difference ≤ 10 g, the same age and physical condition, and divide them into two groups (50% male and female in each group). Create wounds on the back of each rat. In the control group, the wound surface was wrapped with a three-dimensional dressing with an IpR screen-printed electrode. In the experimental group, before wrapping, 2 mL of S. aureus (ATCC 25923) culture solution with a cell density of 100 CFU / mL was inoculated on the wound surface. -1 Complete nutrition was given during the experiment, and the breeding environment was at a temperature of 24 ± 1 °C and a humidity of 55 ± 10%. During this period, continuously record the pH reading results of the IpR screen-printed electrode. During the experiment, regularly draw 100 μL of wound exudate from the liquid storage bottle and measure the IL-6 content level in it by ELISA method. Use the blind experiment method to ask the medical staff participating in the experiment to judge the time point of wound deterioration of the experimental animals by visual inspection based on experience. The results are as Figure 4 shown:

[0087] Figure 4 a The results show that starting from the second day after the infection strain was introduced, the expression level of the wound inflammation indicator IL-6 began to increase significantly, reached the maximum change rate on the 4th day, and approached the highest level observed in the experiment on the 6th day. The synchronous measurement results of the wound surface pH recorded by the IpR screen-printed electrode showed almost the same change trend as the change in the IL-6 level, and broke through the neutral pH level on the 3rd - 4th day, indicating the occurrence of obvious wound infection. And Figure 4b It is judged based on the experience of medical staff. 65% of the staff judged that wound infection occurred on the 6th day, 15% judged that the infection occurred on the 7th day, and only 20% judged that the infection occurred on the 5th day. It can be seen that by making a judgment based on experience, the time when correct wound infection occurs can be 2 - 3 days later than precise quantitative analysis, which will lead to missing valuable early intervention treatment opportunities in clinical practice. The results show that the IpR screen-printed electrode can effectively record the real-time dynamic changes of the pH value of chronic wound surfaces, and the changes can be used to indicate the occurrence of wound infection and inflammation. Rational utilization of it can greatly shorten the cycle for medical staff to detect the occurrence of wound infection and provide valuable time for early intervention of infection.

[0088] Comparative Example 2

[0089] IpR Flexible Electrode for Safe Real-Time Monitoring of pH of VSD Contact Wound Surfaces

[0090] Install the IR flexible electrode described in Example 1 and the IpR flexible electrode in Example 5 into the wound surface contact protection layer of the VSD special three-dimensional dressing respectively. Lead the ends of the electrodes outside the dressing and connect them to the sensing device. Select 10 healthy rats with a body weight difference ≤ 10 g, the same age and physical condition, and divide them into two groups (50% male and female in each group). Create wounds on the back of each rat. In the control group, wrap the wound surface with a three-dimensional dressing with an IR flexible electrode; in the experimental group, wrap the wound surface with a three-dimensional dressing with an IpR flexible electrode. Provide complete nutrition during the experiment, and the breeding environment is at a temperature of 24 ± 1 °C and a humidity of 55 ± 10%. Continuously record the pH reading results of the IR flexible electrode and / or the IpR flexible electrode during this period.

[0091] During the experiment, regularly draw 100 μL of wound exudate from the storage bottle and measure the content levels of IL-4 and IL-13 in it by ELISA method. The changes of the two cytokines during the experimental cycle are as Figure 5As shown: In the monitoring period of 12 consecutive days, the IR electrode caused a significant increase in the IL-4 level in the wound surface after 6 days of wearing, and a significant increase in the IL-13 level occurred on the 12th day of wearing. IL-13 and IL-4 are important Th2-type cytokines and play a key role in allergic reactions. They can stimulate B cells to differentiate into plasma cells and promote these cells to produce IgE antibodies, which is an important step in allergic reactions. In addition, IL-4 can also promote the activation and proliferation of mast cells and basophils, thereby enhancing allergic reactions. The change in its level can directly reflect the intensity of allergic reactions. This shows that conventional iridium-based electrodes as wound-wearing electrode materials can trigger certain allergic reactions and have potential risks. In contrast, no allergic reactions were observed in the wearing test of the IpR electrode, indicating its ideal biocompatibility and suitability as a sensing material for long-term wound-wearing devices directly applied to pH monitoring scenarios that require direct contact with the wound surface.

Claims

1. A pH-sensitive electrode for a wearable pH sensor for diagnosing wound surface status, characterized in that It is prepared by the following method: (1) Use a triethanolamine DMSO solution to adjust the pH of the IrCl4 solution to the level of 2.0 - 6.0, add a DMSO solution of 3-methyl-1,2-cyclopentanedione, mix evenly, and then let it stand in the dark. (2) Heat the mixed solution obtained in step (1) to 75 - 100 °C, add a sodium L-ascorbate solution thereto under stirring conditions, react for 0.2 - 4.5 h, collect the precipitate, namely IrNP, and disperse it in water to obtain an IrNP dispersion. (3) Prepare an aqueous solution of 4,4'-diamino-5,5''-(dithiophen-2-yl)-2,2'-bipyridine, add this solution to the IrNP dispersion under stirring conditions, react at 45 - 70 °C for 12 - 24 h, collect the precipitate after the reaction, redisperse the precipitate in water, and prepare a dispersion system with a mass fraction of 0.05 - 0.5%. (4) Add Ru to the dispersed system obtained in step (3) 3+ The clean base electrode is immersed in the solution, treated at a constant temperature of 40-60°C for 45-80 minutes, then taken out and washed, and then immersed in Ru-containing 3+ The electrode was immersed in an aqueous solution of bipyridine for 60 to 180 minutes to obtain a pH sensitive electrode, which was recorded as IR.

2. The pH-sensitive electrode according to claim 1, wherein: After the IrNP is prepared in step (2), it includes a modification step, specifically dispersing the IrNP in water containing NaOH, D- / L-cysteine and glycerol, and refluxing at 65 - 100 °C for 16 - 48 h to obtain IrNP-pS. Replacing the IrNP dispersion in step (3) with an IrNP-pS dispersion, the electrode IpR can be prepared.

3. The pH-sensitive electrode according to claim 2, wherein: The concentrations of the NaOH, D- / L-cysteine and glycerol in water are 0.15 - 1.0 M: 2 - 60 mM: 0.02 - 0.2 M; The mass fraction of the IrNP in water is 0.05 - 1%.

4. The pH-sensitive electrode according to claim 1, wherein: The concentration of the IrCl4 solution in step (1) is 5 - 15 mM; the concentration of the DMSO solution of 3-methyl-1,2-cyclopentanedione is 0.1 - 0.25 M; the volume ratio of the IrCl4 solution to the DMSO solution of 3-methyl-1,2-cyclopentanedione is 100:20 - 75; The volume ratio of the mixed solution obtained in step (1) to the sodium L-ascorbate solution is 12 - 18:5 - 25.

5. The pH-sensitive electrode according to claim 1, wherein: The mass fraction of the IrNP dispersion in step (2) is 0.01% - 2%; The concentration of the aqueous solution of 4,4'-diamino-5,5''-(dithiophen-2-yl)-2,2'-bipyridine in step (3) is 10 - 20 mM, and its volume ratio to the IrNP dispersion is 2:4 - 12; The mass fraction of the dispersion system in step (3) is 0.1 - 0.5%.

6. The pH-sensitive electrode according to claim 1, wherein: Step (4) For the first time, the Ru-containing 3+ aqueous solution has a Ru 3+ concentration of 1 to 20 mM; The temperature of the first constant temperature treatment in step (4) is 45 - 55 °C; Step (4) The concentration of the second aqueous solution of Ru 3+ is 1 to 20 mM; The concentration of the bipyridine solution in step (4) is 2 - 10 mM.

7. The pH-sensitive electrode according to claim 1, wherein: The concentration of the triethanol in step (1) is 0.1 - 1 M; The temperature for standing still in step (1) is 20 to 35 °C, and the standing time is 12 to 36 h.

8. The pH-sensitive electrode according to claim 1, characterized in that: The substrate electrode in step (4) is at least one of gold, silver, copper, nickel, iridium, platinum, chromium and their alloys, graphite, carbon black, carbon nanotubes, graphene and its derivatives, and indium tin oxide.

9. Use of the pH-sensitive electrode according to any one of claims 1 to 8 in an electrochemically calibration-free pH sensing method, characterized in that comprising the following steps: Detecting the pH of a pH standard buffer solution using an electrode system containing the above-mentioned obtained pH-sensitive electrode and pH-insensitive electrode; The pH indication parameter a.u. obtained by voltammetry is defined as where E f is the peak potential, HWHM is the half-peak half-width potential, ΔI1 and ΔI2 are the peak current intensities corresponding to the pH-sensitive electrode and the pH-insensitive electrode respectively, and a is the electrode constant; Generating a regression curve according to the relationship between the a.u. value and the pH value of the standard buffer solution; Detecting the wound exudate at the wound to be measured using an electrode system composed of the same pH-sensitive electrode and pH-insensitive electrode, and measuring the pH value according to the regression curve; Both the current and potential signals generated by the pH-sensitive electrode will respond to the change of the pH to be measured; The current and potential signals generated by the pH-insensitive electrode do not respond to the change of pH.

10. The application according to claim 9, characterized in that: The pH-insensitive electrode is at least one of gold, silver, copper, nickel, iridium, platinum, chromium and their alloys, graphite, carbon black, carbon nanotubes, graphene and its derivatives, and indium tin oxide, or is prepared by modifying a functional material or in-situ introducing a functional material on the above electrode materials.

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