Enzyme-responsive color change-based bacterial infection self-monitoring dressing, its preparation method and application

Abstract

This invention relates to the field of bacterial monitoring technology, providing a self-monitoring dressing for bacterial infection based on enzyme-responsive color change, its preparation method, and its application. The dressing of this invention comprises a substrate and an enzyme-responsive layer loaded thereon. The enzyme-responsive layer contains a bacterial chromogenic agent and a functional chromogenic aid; the functional chromogenic aid contains sodium dihydrogen phosphate, potassium ferricyanide, and poloxamer. Based on enzyme-responsive color change, this dressing can utilize wound exudate to trigger detection, requiring no additional instruments or liquid reagents. Infection status can be monitored in real-time and intuitively through visible color changes. In particular, the synergistic effect of the functional chromogenic aid significantly improves the detection sensitivity for Gram-positive bacteria, represented by Staphylococcus aureus, achieving simultaneous coverage of multiple common wound pathogens and effectively suppressing false-positive background. It is highly efficient, convenient, and suitable for home monitoring of chronic wound infections.

Description

Technical Field

[0001] This invention relates to the field of bacterial infection self-monitoring dressing technology, specifically to a bacterial infection self-monitoring dressing based on enzyme-responsive color change, its preparation method, and its application. Background Technology

[0002] Bacterial infections of chronic wounds (such as diabetic foot ulcers and pressure ulcers) are a key contributing factor to amputation and even death in patients. Therefore, early and precise treatment is crucial. However, current clinical testing methods and existing rapid testing technologies have significant limitations. Traditional clinical diagnosis relies on swab cultures after removing dressings, which is time-consuming (24-48 hours) and disrupts the wound healing environment. Healthcare workers cannot directly and in real-time assess the infection status. Especially during home wound care, the lack of experience in assessing wound infection can lead to delayed treatment due to failure to promptly identify and implement appropriate anti-infection measures. Extensive prophylactic use of antibiotics based on experience can easily lead to antibiotic abuse. Therefore, developing wound dressings that intelligently respond to the microenvironment of bacterial-infected wounds with color-changing properties is of great significance for wound care both in and out of hospitals.

[0003] Existing chromogenic wound dressings that respond to changes in wound pH have an inherent drawback: a low correlation with bacterial infection. While enzyme-responsive rapid-coloring materials show high correlation with bacterial infection, they suffer from high false-positive rates and low sensitivity against Gram-positive bacteria in practical applications. Commonly used substrates (such as indoleacetic acid) are limited by the thick cell wall barrier of Staphylococcus aureus, the "closed" conformation of lipases, and the solubility limitations of hydrophobic substrates. Conventional surfactants are also unable to effectively induce color development, resulting in a detection threshold (10⁻⁶). 8 The CFU / mL level was far higher than the clinically required early warning standard (10). 6 (CFU / mL), which is insufficient for effective early screening of pathogens. Summary of the Invention

[0004] Therefore, it is necessary to provide a bacterial infection self-monitoring dressing based on enzyme-responsive color change, its preparation method and application, which can overcome the defects of existing technologies and achieve high sensitivity, low false positives, and intuitive monitoring of wound bacterial infection.

[0005] The present invention adopts the following technical solution: The present invention provides a bacterial infection self-monitoring dressing based on enzyme-responsive color change, the dressing comprising a substrate and an enzyme-responsive layer loaded on the substrate; The enzyme response layer contains a bacterial chromogenic agent and a functional chromogenic aid; the bacterial chromogenic agent is selected from indole acetate or its halogenated derivatives (e.g., 5-bromo-4-chloro-3-indole acetate) or p-nitrobenzene ester chromogenic agents. The functional colorimetric agent comprises at least sodium dihydrogen phosphate and potassium ferricyanide, and the preferred mass ratio of bacterial colorimetric agent, sodium dihydrogen phosphate, and potassium ferricyanide is 1:(6~12):3.

[0006] The functional colorimetric agent more preferably contains poloxamer, and the mass ratio of bacterial colorimetric agent, sodium dihydrogen phosphate, potassium ferricyanide and poloxamer is 1:12:3:20.

[0007] In some preferred embodiments, the thickness of the enzyme-responsive layer is 0.1~0.5 mm, preferably 0.2 mm. The dressing itself is white or light pink in color to facilitate observation of color changes.

[0008] In some preferred embodiments, the substrate is selected from cotton fabric, silk fabric, hydrophilic synthetic fiber fabric, or polyurethane foam dressing.

[0009] This invention also provides a method for preparing a bacterial infection self-monitoring dressing based on enzyme-responsive color change, comprising the following steps: The functional color-developing agent is dissolved in water, and the pH is adjusted to 6 to obtain a functional color-developing agent solution; the substrate is immersed in the functional color-developing agent solution, removed and dried to obtain a fiber substrate loaded with the functional color-developing agent. The bacterial chromogenic agent was dissolved in anhydrous ethanol to obtain a chromogenic induction solution; the substrate loaded with the functional chromogenic aid was immersed in the chromogenic induction solution to obtain an enzyme-responsive loaded material. The enzyme-responsive loading material is dried to remove the solvent and then encapsulated to obtain the bacterial infection self-monitoring dressing.

[0010] In some preferred embodiments, the impregnation process is a solution impregnation method; the drying is freeze drying or low-temperature vacuum drying to avoid high temperature damaging the substrate activity.

[0011] In some preferred embodiments, the residual ethanol content in the dressing is <1%.

[0012] This invention further provides the application of the above-mentioned bacterial infection monitoring dressing in the preparation of medical devices for monitoring bacterial infections in wounds. The bacterial infection includes, but is not limited to, Pseudomonas aeruginosa, Escherichia coli, or Staphylococcus aureus infections, and the wound is accompanied by fluid exudation.

[0013] Compared with the prior art, the core technical advantages and beneficial effects of this invention are as follows: 1. High Sensitivity and Broad Spectrum: The dressing of this invention constructs a unique hydrophilic-hydrophobic interface environment through the synergistic effect of specific types and ratios of functional chromogenic auxiliaries. This environment can effectively penetrate the thick cell walls of Gram-positive bacteria such as Staphylococcus aureus, inducing their lipase structures to "open," thereby significantly improving the detection sensitivity for Gram-positive bacteria. Simultaneously, it is also effective against Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli, achieving simultaneous coverage of multiple common wound pathogens. Potassium ferricyanide also plays a catalytic role in the colorimetric reaction. Experiments show that it is effective at concentrations ≥10... 3 The target bacteria at a concentration of CFU / mL can produce a visible color change, meeting the needs for early clinical warning.

[0014] 2. Low false positive rate: This invention utilizes sodium dihydrogen phosphate to construct a solid, weakly acidic buffer microenvironment (buffering the local pH to approximately 6.0). When the dressing is only moistened by sterile wound exudate (pH approximately 7.4), this buffer environment effectively inhibits the spontaneous chemical hydrolysis of the indole ester substrate, thereby eliminating the false positive blue background. Substrate hydrolysis is catalyzed only in the presence of bacterial esterases.

[0015] 3. Convenience and Safety: This invention's dressing requires no testing instruments or added liquid reagents; detection is triggered solely by the wound's own exudate, with results readily visible to the naked eye. It is highly efficient and convenient, especially suitable for home monitoring of chronic wound infections. The preparation process uses anhydrous ethanol solvent, which offers better permeability than aqueous impregnation processes and can be completely removed through freeze-drying, resulting in extremely low ethanol residue (<1%) and excellent biocompatibility. Attached Figure Description

[0016] Figure 1 The color changes of the blank sample under different test conditions in Example 1 are shown.

[0017] Figure 2 This is a comparison chart showing the color development of the functional chromogenic agent in Example 2 on different concentrations of Staphylococcus aureus (S), Pseudomonas aeruginosa (P), and Escherichia coli (E).

[0018] Figure 3 This is a comparison chart of the color development of the non-functional chromogenic agent in Example 2 for detecting Staphylococcus aureus (S), Pseudomonas aeruginosa (P), and Escherichia coli (E) at different concentrations.

[0019] Figure 4 The different proportions of the chromogenic dressings in Example 4 were used to treat three bacteria at 10... 6Comparison of colorimetric effects after 12 hours of detection at CFU / mL concentration; where "1" corresponds to "Group 1" of the mass ratio experiment of indole acetate, sodium dihydrogen phosphate, and potassium ferricyanide, "2" corresponds to "Group 2" of the mass ratio experiment of indole acetate, sodium dihydrogen phosphate, and potassium ferricyanide, "3" corresponds to "Group 3" of the mass ratio experiment of indole acetate, sodium dihydrogen phosphate, and potassium ferricyanide, and so on, "6" corresponds to "Group 6" of the mass ratio experiment of indole acetate, sodium dihydrogen phosphate, and potassium ferricyanide. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. The following embodiments are only used to illustrate the present invention, and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0021] Example 1: Preparation and Performance of Dressing This embodiment provides a dressing preparation method, including the following steps: (1) Preparation of functional colorimetric additive loading.

[0022] Dissolve 1.2g sodium dihydrogen phosphate, 0.3g potassium ferricyanide, and 2g poloxamer in 100mL of deionized water, and adjust the pH to 6 with 0.1M sodium hydroxide solution and / or dilute hydrochloric acid solution to obtain a functional colorimetric auxiliary solution.

[0023] At room temperature, immerse the cotton fabric (basic information: spunlace cotton-based nonwoven fabric) in a functional color-developing agent solution for 5-10 minutes, then remove and freeze-dry.

[0024] (2) Preparation of colorimetric load.

[0025] Dissolve 0.1 g of indoleacetic acid in 100 mL of anhydrous ethanol to obtain a colorimetric induction solution.

[0026] The cotton fabric dried in step (1) above is immersed in the color-developing induction solution at room temperature for 5-10 minutes to obtain the color-developing load.

[0027] (3) Prepare the finished dressing.

[0028] Take out the color-developing load (fabric) from step (2), dry it at a low temperature of 40°C under vacuum until constant weight, ensure that the residual ethanol content is <1%, and package it to obtain the finished dressing.

[0029] Performance testing: The dressings prepared in this embodiment were added dropwise to sterile deionized water (ddH2O), PBS buffer (pH 7.2), and aqueous solutions of different pH values ​​(4, 7, and 10), and incubated at 37°C for 12 hours.

[0030] The color change results of the dressing in this embodiment under different incubation environments are as follows: Figure 1 As shown.

[0031] Depend on Figure 1 It can be seen that all groups of dressings remained white after 12 hours of incubation, indicating that the dressings remained stable under different environmental conditions and pH levels, with no false positives.

[0032] Example 2: Effect of functional color-developing additives on the color development performance of dressings This embodiment aims to investigate the effect of functional chromogenic dressings on the color development effect under the action of different concentrations of bacteria.

[0033] 1. Preparation of colorimetric induction solution: (1) Induction solution containing functional colorimetric auxiliaries (experimental group): Indoleacetic acid (98% purity) was dissolved in anhydrous ethanol to prepare a 2% stock solution. Then, sodium dihydrogen phosphate (99% purity), potassium ferricyanide (99.5% purity), and poloxamer were added sequentially to the stock solution, and the pH was adjusted to 6.0 with 0.1M sodium hydroxide solution and / or dilute hydrochloric acid solution to obtain a colorimetric induction solution containing 0.1% (w / v) indoleacetic acid, 1.2% (w / v) sodium dihydrogen phosphate, 0.3% (w / v) potassium ferricyanide, and 2% (w / v) poloxamer.

[0034] (2) Induction solution without functional colorimetric additives (control group): Indoleacetic acid (98% purity) was dissolved in anhydrous ethanol to prepare a 2% stock solution. This stock solution was then diluted with anhydrous ethanol to obtain a colorimetric induction solution containing only 0.1% (w / v) indoleacetic acid, free of sodium dihydrogen phosphate, potassium ferricyanide, and poloxamer.

[0035] 2. Preparation of dressing samples: At room temperature, pure cotton nonwoven fabric was immersed in the color-inducing solution of the experimental group and the control group for 5-10 minutes, respectively. After removal, it was freeze-dried (with the same parameters and conditions as in Example 1), and cut into circular pieces with a diameter of 1.5 cm, which were denoted as follows: (1) Experimental dressing: loaded with indoleacetic acid ester and functional color-developing agents (sodium dihydrogen phosphate, potassium ferricyanide, poloxamer).

[0036] (2) Control group dressing: only loaded with indoleacetic acid ester, without functional color-developing additives.

[0037] 3. Bacterial detection experiment: Staphylococcus aureus (S), Pseudomonas aeruginosa (P), and Escherichia coli (E) were diluted to 10 μL with sterile PBS buffer. 9 CFU / mL, 10 8 CFU / mL, 10 7 CFU / mL, 10 6 CFU / mL, 10 5 CFU / mL and 10 4 CFU / mL.

[0038] The dressing discs for the experimental and control groups were placed in 24-well plates, and 100 μL of bacterial suspensions of different concentrations were added to each well. The bacterial load of the dressings was 10... 8 10 7 10 6 10 5 10 4 10 3 CFU / mL. Incubate the 24-well plate in a 37°C incubator for 12 hours, and observe and record the color changes.

[0039] 4. Experimental Results: (1) The color development results of the experimental group dressing (including functional color-developing additives) are as follows: Figure 2 As shown: After 12 hours of incubation, the monitored concentration of Staphylococcus aureus (S) in the dressing containing the functional chromogenic agent was 10. 7 The monitored concentration of CFU / mL for Pseudomonas aeruginosa (P) and Escherichia coli (E) reached 10. 3 The concentration of bacteria was CFU / mL, and the colorimetric intensity gradually increased with increasing bacterial concentration.

[0040] This indicates that using a combination of sodium dihydrogen phosphate, potassium ferricyanide, and poloxamer as a functional colorimetric aid can effectively synergistically activate the colorimetric reaction and achieve the desired effect on 10... 3 Effective detection of Pseudomonas aeruginosa (P) and Escherichia coli (E) infections at concentrations of CFU / mL and above, meeting the needs of early clinical warning.

[0041] (2) The color development results of the control group dressing (without functional color-developing additives) are as follows: Figure 3 As shown: Dressings without functional color-developing agents are only detected at high concentrations (10). 7 10 8 The slight color change (CFU / mL) indicates that dressings loaded with indoleacetic acid alone cannot effectively respond to bacterial esterases under current conditions, further demonstrating the indispensability of functional chromogenic aids for achieving enzyme-responsive color changes in dressings.

[0042] Example 3: Effects of different synergists on the detection efficacy of Staphylococcus aureus Referring to the method and steps of Example 2, this example investigates the effect of dressings containing indoleacetic acid chromogenic induction solutions with different synergists, such as CaCl2, Triton X-100, and Poloxamer 407, on the chromogenic monitoring of Staphylococcus aureus (S).

[0043] The colorimetric induction solution containing synergist doleacetic acid ester was mixed with 10 7 Mix CFU / mL Staphylococcus aureus bacterial suspension at a ratio of 10:1 (v / v), incubate at 37°C for 12 h, and observe the color change.

[0044] The types and concentrations of synergists and their colorimetric monitoring effects are shown in the table below.

[0045] Statistical Table of Synergist Types and Concentrations and Their Effects on Colorimetric Monitoring of Staphylococcus aureus As can be seen from the table above: Within the tested concentration range, indoleacetic acid chromogenic induction solutions containing 2% to 8% (w / v) of poloxamer (Poloxamer 407) effectively promoted the color development (turning blue) of Staphylococcus aureus detection, while indoleacetic acid chromogenic induction solutions with added CaCl2 or Triton X-100 did not show any color development of Staphylococcus aureus detection at any tested concentration.

[0046] The results show that: Poloxamer is a key synergist that enhances the colorimetric response of indoleacetic acid substrates to Staphylococcus aureus.

[0047] Example 4 Referring to the method and steps of Example 2, this example investigates the effect of different proportions of indoleacetic acid, sodium dihydrogen phosphate, and potassium ferricyanide in the colorimetric induction solution and its dressing on bacteria (10... 6 The impact of CFU / mL) monitoring effectiveness.

[0048] The statistical results are shown in the table below. Figure 4 .

[0049] Statistical analysis of the effects of different component ratios in chromogenic induction solutions and dressings on the effectiveness of bacterial chromogenic monitoring The test results show that: When the mass ratio of indoleacetic acid, sodium dihydrogen phosphate, and potassium ferricyanide is 1:(6~12):3, colorimetric detection of the three bacteria can be achieved. Among them, the optimal ratio is 1:12:3 (Group 1), which shows good to excellent detection sensitivity and the best overall performance for all three bacteria.

[0050] Beyond the examples mentioned above, extensive experimental research has revealed that: 1. This invention essentially provides a medical dressing for visually monitoring wound infection, comprising a medical substrate and an enzyme-responsive layer (solid functional coating) loaded on the substrate. The coating contains an enzyme-responsive chromogenic substrate (selected from indoleacetic acid ester and its halogenated derivatives, p-nitrophenyl ester chromogenic agents), sodium dihydrogen phosphate, and potassium ferricyanide, and preferably contains poloxamer series synergists. After contact with bacterial infection exudate, the dressing can respond to concentrations ≥10 within 12 hours. 6 Staphylococcus aureus CFU / mL, ≥10 3 CFU / mL of Escherichia coli and Pseudomonas aeruginosa produce visible color changes, making it particularly suitable for home monitoring of wound infections caused by chronic diseases.

[0051] 2. The dressing of the present invention especially needs to control the mass ratio of enzyme-responsive chromogenic substrate, sodium dihydrogen phosphate, and potassium ferricyanide to be within the range of 1:(6~12):3. When bacterial infection occurs in the wound, the esterase secreted by the bacteria diffuses into the dressing layer with the exudate. It is activated by the combined environmental conditions of poloxamer, sodium dihydrogen phosphate, and potassium ferricyanide and hydrolyzes the substrate, and the dressing turns blue-green at the corresponding position. If there is no bacterial infection, even if it is wetted by exudate, the dressing can always maintain its original white or light pink color due to the presence of the weakly acidic buffer system of poloxamer-sodium dihydrogen phosphate.

[0052] 3. In the dressing preparation process of this invention, anhydrous ethanol is used as a solvent. In the final product, the anhydrous ethanol is completely evaporated or the residual amount is extremely low (<1%), ensuring no interference with the color development results. The content of poloxamer synergist is preferably controlled within the range of 8%, which has excellent biocompatibility and is gentler on the skin and wound tissue.

[0053] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A bacterial infection self-monitoring dressing based on enzyme-responsive color change, characterized in that, The dressing includes a substrate and an enzyme-responsive layer loaded on the substrate; The enzyme response layer contains bacterial chromogenic agents and functional chromogenic aids; The bacterial chromogenic agent is selected from indoleacetic acid ester or its halogenated derivatives, and p-nitrophenyl ester chromogenic agents; The functional color-developing agent contains sodium dihydrogen phosphate and potassium ferricyanide.

2. The bacterial infection self-monitoring dressing based on enzyme-responsive color change according to claim 1, characterized in that, The mass ratio of bacterial chromogenic reagent, sodium dihydrogen phosphate, and potassium ferricyanide is 1:(6~12):

3.

3. The bacterial infection self-monitoring dressing based on enzyme-responsive color change according to claim 1 or 2, characterized in that, The functional colorimetric agent also includes poloxamer, and the mass ratio of bacterial colorimetric agent, sodium dihydrogen phosphate, potassium ferricyanide, and poloxamer is 1:(6~12):3:

20.

4. The bacterial infection self-monitoring dressing based on enzyme-responsive color change according to claim 1 or 2, characterized in that, The thickness of the enzyme-responsive layer is 0.1~0.5 mm, and the dressing body is white or light pink.

5. The bacterial infection self-monitoring dressing based on enzyme-responsive color change according to claim 1 or 2, characterized in that, The substrate is selected from cotton fabric, silk fabric, hydrophilic chemical fiber fabric or polyurethane foam dressing.

6. The method for preparing the enzyme-responsive color change-based bacterial infection self-monitoring dressing according to any one of claims 1 to 5, characterized in that, Includes the following steps: A functional color-developing agent is dissolved in water, and the pH is adjusted to 6 to prepare a functional color-developing agent solution. The substrate is immersed in the functional color-developing agent solution, removed and dried to obtain a substrate loaded with the functional color-developing agent. A bacterial chromogenic agent is dissolved in anhydrous ethanol to prepare a chromogenic induction solution; the substrate loaded with the functional chromogenic agent is immersed in the chromogenic induction solution to prepare an enzyme-responsive loading material. The enzyme-responsive loading material is dried to remove the solvent and then encapsulated to obtain the dressing.

7. The method for preparing the enzyme-responsive color change-based bacterial infection self-monitoring dressing according to claim 6, characterized in that, The drying process is freeze drying or low-temperature vacuum drying.

8. The method for preparing the bacterial infection self-monitoring dressing based on enzyme-responsive color change according to claim 6, characterized in that, The residual ethanol content in the dressing is <1%.

9. The use of the enzyme-responsive color-changing bacterial infection self-monitoring dressing according to any one of claims 1 to 5 in the preparation of a medical device for monitoring bacterial infection in wounds.

10. The application according to claim 9, characterized in that, The wound infection is caused by Pseudomonas aeruginosa, Escherichia coli, or Staphylococcus aureus, and is accompanied by fluid exudation.

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