Functionalized partition detecting hydrogel, its preparation method and application

Abstract

This invention discloses a functionalized zoned detection hydrogel, its preparation method, and its applications. The hydrogel is divided into two zones: Zone I is used to detect hydrogen peroxide in reactive oxygen species (ROS) produced by bacteria, and Zone II is used to detect enzymes produced by bacteria. Zone I uses egg white-gold nanoclusters as the functional detection center, obtained by freezing and thawing egg white-gold nanoclusters with polyethylene glycol. The egg white-gold nanoclusters are obtained by reducing tetrachloroauric acid using egg white and cellulose nanocrystals as protective ligands. Zone II uses bovine serum albumin (BSA)-gold nanoclusters as the functional detection center, obtained by freezing and thawing BSA-gold nanoclusters with polyethylene glycol. The BSA-gold nanoclusters are obtained by reducing tetrachloroauric acid using BSA and cellulose nanocrystals as protective ligands. This hydrogel simultaneously achieves zoned visualization and high-sensitivity detection of hydrogen peroxide and enzymes in ROS produced by bacteria at wound sites, and can be applied to monitor the degree of wound infection.

Description

Technical Field

[0001] This invention relates to the field of medical dressings, specifically to a functionalized zone detection hydrogel, its preparation method, and its application. Background Technology

[0002] When the skin is injured, macrophages in the body repair damaged tissue without any treatment. Wounds are usually irregular in shape, and infecting bacteria adhere to them. The extracellular polymeric substances produced by the adhering bacteria and their cells form a bacterial biofilm. Within this biofilm, bacteria continuously stratify and differentiate, increasing in number and antibiotic resistance, thus competing for nutrients needed by normal cells. This leads to reduced metabolic activity in normal cells and slows wound healing. Therefore, chronic wound healing cannot rely solely on the body's own healing abilities; wound dressings are needed to promote healing and prevent external pathogens from contacting the wound. However, the wound healing process is complex and variable. Using dressings to promote wound healing while simultaneously monitoring the wound provides new insights into health monitoring, wound treatment, and on-demand care.

[0003] To detect wound condition, bacterial-secreted enzymes can serve as physicochemical markers. ROS overload generated by bacterial-infected wounds can induce oxidative stress and endothelial dysfunction, restricting angiogenesis and further leading to chronic wound formation and stagnation of the wound healing process. Therefore, detecting reactive oxygen species (ROS), which are closely related to the state of infected wounds, can provide feasible suggestions for wound treatment and effectively reduce the delay between infection identification and treatment. Developing antibacterial wound dressings capable of real-time monitoring of bacteria and bacterial infection during wound healing, and providing effective treatment based on the results, is of significant research and development value for the visualization and identification of bacteria. Summary of the Invention

[0004] This invention provides a functionalized zoned detection hydrogel that enables zoned visualization and high-sensitivity detection of hydrogen peroxide and alkaline phosphatase in reactive oxygen species produced by bacteria at wound sites, and can be applied to monitor the degree of infection in wounds.

[0005] The above-mentioned objectives of the present invention are achieved through the following technical solutions:

[0006] A functionalized zone detection hydrogel, wherein the functionalized zone detection hydrogel is divided into a first zone and a second zone, the first zone is used to detect hydrogen peroxide in reactive oxygen species produced by bacteria, and the second zone is used to detect enzymes produced by bacteria;

[0007] The first region is obtained by freezing and thawing egg white-gold nanoclusters with polyethylene glycol, using egg white-gold nanoclusters as the functional detection center; the egg white-gold nanoclusters are obtained by reducing tetrachloroauric acid with egg white and cellulose nanocrystals as protective ligands.

[0008] The second region is obtained by freezing and thawing bovine serum albumin-gold nanoclusters with polyethylene glycol, using bovine serum albumin-gold nanoclusters as the functional detection center; the bovine serum albumin-gold nanoclusters are obtained by reducing tetrachloroauric acid with bovine serum albumin and cellulose nanocrystals as protective ligands.

[0009] The functionalized regional detection hydrogel of the present invention is prepared by the following method:

[0010] S1. Preparation of egg white-gold nanoclusters: Tetrachloroauric acid solution was added to a cellulose nanocrystal dispersion, followed by egg white. The mixture was reacted at 30–120°C for 3–48 h to obtain egg white-gold nanoclusters. The concentration of the tetrachloroauric acid solution was 5.0–20.0 mmol / L, the mass percentage of the cellulose nanocrystal dispersion was 1–3 wt%, and the corresponding volume ratio of the tetrachloroauric acid solution to the cellulose nanocrystal dispersion was 1:1–2. The volume ratio of the egg white to the cellulose nanocrystal dispersion was also 1:1–2.

[0011] S2. Preparation of bovine serum albumin-gold nanoclusters: Tetrachloroauric acid solution was added to a cellulose nanocrystal dispersion, followed by the addition of bovine serum albumin. The mixture was reacted at 30–120°C for 3–48 h to obtain bovine serum albumin-gold nanoclusters. The concentration of the tetrachloroauric acid solution was 0.1–50.0 mmol / L, the mass percentage of the cellulose nanocrystal dispersion was 1–3 wt%, and the corresponding volume ratio of the tetrachloroauric acid solution to the cellulose nanocrystal dispersion was 1:1–3. The volume ratio of the bovine serum albumin to the cellulose nanocrystal dispersion was also 1:1–3.

[0012] S3. Preparation of Functionalized Zone Detection Hydrogel: The egg white-gold nanoclusters obtained in step S1 were added to a polyethylene glycol solution with a mass concentration of 1-50 wt% at a volume ratio of 1-5:1 and stirred for 4-48 hours to obtain an egg white-gold nanocluster mixture; the bovine serum albumin-gold nanoclusters obtained in step S2 were added to a polyethylene glycol solution with a mass concentration of 1-50 wt% at a volume ratio of 1-5:1 and stirred for 4-48 hours to obtain a bovine serum albumin-gold nanocluster mixture; the obtained egg white-gold nanocluster mixture was poured into a zone one mold and the obtained bovine serum albumin-gold nanocluster mixture was poured into a zone two mold and placed in the freezer layer of a refrigerator for 3-5 cycles of freeze-thaw operation; the functionalized zone one detection hydrogel and the functionalized zone two detection hydrogel were combined together, and the hydrogels of the two zones were tightly adhered together by their own viscosity to obtain the functionalized zone detection hydrogel.

[0013] The functionalized zone detection hydrogel of this invention can be used to monitor the degree of infection in wounds. One zone of the functionalized zone detection hydrogel is used to detect hydrogen peroxide in reactive oxygen species produced by bacteria, with a detection sensitivity of up to 10 for hydrogen peroxide. - 5 The detection process involves functionalizing the hydrogel in zone one, where the red fluorescence is rapidly quenched. A colorimetric method is used to compare the intensity of the red fluorescence to indicate the hydrogen peroxide content. Zone two is used to detect enzymes produced by Gram-negative bacteria, with a sensitivity of 0.01–3000 U / L for alkaline phosphatase secreted by Gram-negative bacteria. During detection, the blue fluorescence of the hydrogel in zone two is quenched, and a colorimetric method is used to compare the intensity of the blue fluorescence to indicate the enzyme content.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) This invention uses protein and cellulose nanocrystals as protective ligands to reduce tetrachloroauric acid to obtain gold nanoclusters, which are then mixed with polyethylene glycol solution, poured into a partitioned mold, and subjected to repeated freeze-thaw cycles to prepare a flexible hydrogel that displays the color of hydrogen peroxide and alkaline phosphatase in reactive oxygen species. This hydrogel can simultaneously achieve visual detection of hydrogen peroxide and alkaline phosphatase, with high detection sensitivity; the detection sensitivity for hydrogen peroxide and alkaline phosphatase can reach 10, respectively. -5 0.01 mol / L and 0.01 U / L can be used to monitor the degree of infection in wounds.

[0016] (2) In this invention, protein and cellulose nanocrystals are used as protective ligands to reduce tetrachloroauric acid to obtain gold nanoclusters. The dispersibility of cellulose nanocrystals is used to increase the stability of gold nanoclusters and prolong the fluorescence lifetime during the detection process of gold nanoclusters.

[0017] (3) After preparing gold nanoclusters, the present invention obtains a flexible hydrogel by adjusting the concentration ratio of polyethylene glycol through freeze-thaw process. The flexible hydrogel can adapt to wounds, adhere to the skin surface without falling off, and adapt to the irregular shape of wounds.

[0018] (4) After preparing gold nanoclusters, the present invention controls the concentration ratio of polyethylene glycol and obtains a thin sheet-like hydrogel with a certain thickness by freeze-thaw method. This ensures that the detection sensitivity of gold nanoclusters to hydrogen peroxide and alkaline phosphatase is guaranteed and does not affect their application. At the same time, the thin sheet-like hydrogel is more convenient for visualization and observation. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the structure of a functionalized partitioned detection hydrogel according to the present invention.

[0020] Explanation of markings in the diagram: 1 represents zone one, and 2 represents zone two. Detailed Implementation

[0021] As attached Figure 1 As shown, the functionalized zone detection hydrogel of the present invention is divided into zone 1 and zone 2. Zone 1 is used to detect hydrogen peroxide in reactive oxygen species produced by bacteria, and zone 2 is used to detect enzymes produced by bacteria.

[0022] The first region is obtained by freezing and thawing egg white-gold nanoclusters with polyethylene glycol, using egg white-gold nanoclusters as the functional detection center; the egg white-gold nanoclusters are obtained by reducing tetrachloroauric acid with egg white and cellulose nanocrystals as protective ligands.

[0023] The second region is obtained by freezing and thawing bovine serum albumin-gold nanoclusters with polyethylene glycol, using bovine serum albumin-gold nanoclusters as the functional detection center; the bovine serum albumin-gold nanoclusters are obtained by reducing tetrachloroauric acid with bovine serum albumin and cellulose nanocrystals as protective ligands.

[0024] Example 1

[0025] The functionalized regional detection hydrogel of the present invention is prepared by the following steps:

[0026] S1. Preparation of egg white-gold nanoclusters: 5 mL of 10.0 mmol / L tetrachloroauric acid solution was added to 5 mL of 1.37 wt% cellulose nanocrystal dispersion, and then 5 mL of egg white was added. The mixture was reacted at 30 °C for 3 h to obtain egg white-gold nanoclusters, which were then stored in the dark at 4 °C.

[0027] S2. Preparation of bovine serum albumin-gold nanoclusters: 5 mL of 10.0 mmol / L tetrachloroauric acid solution was added to 5 mL of 1.37 wt% cellulose nanocrystal dispersion, and then 5 mL of bovine serum albumin was added. The mixture was reacted at 30 °C for 3 h to obtain bovine serum albumin-gold nanoclusters, which were then stored in the dark at 4 °C.

[0028] S3. Preparation of Functionalized Zone Detection Hydrogel: The egg white-gold nanoclusters obtained in step S1 were added to a 1wt% polyethylene glycol solution at a volume ratio of 1:1 and stirred vigorously for 4 hours to obtain an egg white-gold nanocluster mixture; the bovine serum albumin-gold nanoclusters obtained in step S2 were added to a 1wt% polyethylene glycol solution at a volume ratio of 1:1 and stirred vigorously for 4 hours to obtain a bovine serum albumin-gold nanocluster mixture; the obtained egg white-gold nanocluster mixture was poured into a zone one mold, and the obtained bovine serum albumin-gold nanocluster mixture was poured into a zone two mold and placed in the freezer layer of a refrigerator for 3 cycles of freeze-thaw operation; the prepared functionalized zone one detection hydrogel and functionalized zone two detection hydrogel were combined together, and the hydrogels of the two zones adhered tightly together due to their own viscosity to obtain the functionalized zone detection hydrogel.

[0029] Example 2

[0030] The functionalized regional detection hydrogel of the present invention is prepared by the following steps:

[0031] S1. Preparation of egg white-gold nanoclusters: 5 mL of 5.0 mmol / L tetrachloroauric acid solution was dissolved in 10 mL of 2.1 wt% cellulose nanocrystal dispersion, and then 5 mL of egg white was added. The mixture was reacted at 37 °C for 24 h to obtain egg white-gold nanoclusters, which were then stored in the dark at 4 °C.

[0032] S2. Preparation of bovine serum albumin-gold nanoclusters: 5 mmol / L tetrachloroauric acid solution was dissolved in 10 mL of 2.1 wt% cellulose nanocrystal dispersion, and then 5 mL of bovine serum albumin was added. The mixture was reacted at 37 °C for 24 h to obtain bovine serum albumin-gold nanoclusters, which were then stored in the dark at 4 °C.

[0033] S3. Preparation of Functionalized Zone Detection Hydrogel: The egg white-gold nanoclusters obtained in step S1 were added to a 25wt% polyethylene glycol solution at a volume ratio of 3:1 and stirred vigorously for 24 hours to obtain an egg white-gold nanocluster mixture; the bovine serum albumin-gold nanoclusters obtained in step S2 were added to a 25wt% polyethylene glycol solution at a volume ratio of 3:1 and stirred vigorously for 24 hours to obtain a bovine serum albumin-gold nanocluster mixture; the obtained egg white-gold nanocluster mixture was poured into a zone one mold and the obtained bovine serum albumin-gold nanocluster mixture was poured into a zone two mold and placed in the freezer layer of a refrigerator for three cycles of freeze-thaw operation; the prepared functionalized zone one detection hydrogel and functionalized zone two detection hydrogel were combined together, and the hydrogels of the two zones adhered tightly together due to their own viscosity to obtain the functionalized zone detection hydrogel.

[0034] Example 3

[0035] The functionalized regional detection hydrogel of the present invention is prepared by the following steps:

[0036] S1. Preparation of egg white-gold nanoclusters: 10 mL of 20.0 mmol / L tetrachloroauric acid solution was dissolved in 15 mL of 3.0 wt% cellulose nanocrystal dispersion, and then 10 mL of egg white was added. The mixture was reacted at 120 °C for 48 h to obtain egg white-gold nanoclusters, which were then stored in the dark at 4 °C.

[0037] S2. Preparation of bovine serum albumin-gold nanoclusters: 10 mL of 20 mmol / L tetrachloroauric acid solution was dissolved in 15 mL of 3.0 wt% cellulose nanocrystal dispersion, and then 10 mL of bovine serum albumin was added. The mixture was reacted at 120 °C for 24 h to obtain bovine serum albumin-gold nanoclusters, which were then stored in the dark at 4 °C.

[0038] S3. Preparation of Functionalized Zone Detection Hydrogel: The egg white-gold nanoclusters obtained in step S1 were added to a 50wt% polyethylene glycol solution at a volume ratio of 5:1 and stirred vigorously for 48 hours to obtain an egg white-gold nanocluster mixture; the bovine serum albumin-gold nanoclusters obtained in step S2 were added to a 50wt% polyethylene glycol solution at a volume ratio of 5:1 and stirred vigorously for 48 hours to obtain a bovine serum albumin-gold nanocluster mixture; the obtained egg white-gold nanocluster mixture was poured into a zone one mold and the obtained bovine serum albumin-gold nanocluster mixture was poured into a zone two mold and placed in the freezer layer of a refrigerator for three cycles of freeze-thaw operation; the prepared functionalized zone one detection hydrogel and functionalized zone two detection hydrogel were combined together, and the hydrogels of the two zones adhered tightly together due to their own viscosity to obtain the functionalized zone detection hydrogel.

[0039] Performance tests of the gold nanoclusters and functionalized partitioned detection hydrogels prepared in Examples 1, 2, and 3

[0040] ① Prepare concentrations of 10 -5 mol / L, 5*10 -5 mol / L, 10 -4 mol / L, 2*10 -4 mol / L, 5*10 -4 mol / L, 10 -3 mol / L, 3*10 -3 mol / L, 5*10 -3 mol / L, 7*10 -3 mol / L, 9*10 -3 mol / L, 1.2*10 -2 mol / L, 1.5*10 - 2 mol / L, 2*10 -2 mol / L, 3*10 -2 A certain amount of the egg white-gold nanoclusters prepared in Examples 1-3 were taken from a mol / L ·OH solution and added dropwise to the ·OH solution prepared above. The fluorescence changes of the ·OH solution with added egg white-gold nanoclusters were recorded by fluorescence spectroscopy, and the solution was photographed at the same time. The original egg white-gold nanocluster solution was set as the reference sample.

[0041] Results: The egg white-gold nanocluster solutions prepared in Examples 1-3 exhibited red fluorescence under ultraviolet light. When added to ·OH solution, the color of the mixture gradually lightened from red to light red as the concentration increased, and finally the fluorescence disappeared. The fluorescence spectrum recorded the corresponding fluorescence intensity gradually decreased as the concentration increased.

[0042] ② Prepare alkaline phosphatase solutions with concentrations of 0.01 U / L, 0.05 U / L, 0.1 U / L, 0.5 U / L, 1.0 U / L, 5.0 U / L, 10.0 U / L, 50.0 U / L, 100.0 U / L, 300.0 U / L, 500.0 U / L, 1000.0 U / L, 1500.0 U / L, 2000.0 U / L, 2500.0 U / L, and 3000.0 U / L, respectively. Take a certain amount of bovine serum albumin-gold nanoclusters prepared in Examples 1-3 and add an equal amount to the alkaline phosphatase solutions prepared above. Record the corresponding fluorescence changes of the alkaline phosphatase solutions with added bovine serum albumin-gold nanoclusters using fluorescence spectroscopy, and photograph the solutions simultaneously. Set the originally prepared bovine serum albumin-gold nanocluster solution as a reference sample.

[0043] Results: The bovine serum albumin-gold nanoclusters solutions prepared in Examples 1-3 exhibited blue fluorescence under ultraviolet light. When added to alkaline phosphatase solution, the color of the mixture gradually lightened from blue (from egg white to gold nanoclusters) as the concentration increased, and finally the fluorescence disappeared. The fluorescence spectrum recorded the corresponding fluorescence intensity gradually decreased as the concentration increased.

[0044] ③ Prepare concentrations of 10. -5 mol / L, 5*10 -5 mol / L, 10 -4 mol / L, 2*10 -4 mol / L, 5*10 -4 mol / L, 10 -3 mol / L, 3*10 -3 mol / L, 5*10 -3 mol / L, 7*10 -3 mol / L, 9*10 -3 mol / L, 1.2*10 -2 mol / L, 1.5*10 - 2 mol / L, 2*10 -2 mol / L, 3*10 -2 Equal volumes of the functionalized zone-detection hydrogels prepared in Examples 1-3 were immersed in the prepared ·OH solution at mol / L. The hydrogels were then irradiated with a UV lamp while photographing was performed. The originally prepared functionalized zone-detection hydrogel was used as a reference sample.

[0045] Results: The functionalized one-zone detection hydrogels prepared in Examples 1-3 emitted red fluorescence under ultraviolet light. When immersed in OH solution, the color of the functionalized one-zone detection hydrogel gradually lightened with increasing concentration, and finally the fluorescence disappeared.

[0046] ④ Prepare alkaline phosphatase solutions with concentrations of 0.01 U / L, 0.05 U / L, 0.1 U / L, 0.5 U / L, 1.0 U / L, 5.0 U / L, 10.0 U / L, 50.0 U / L, 100.0 U / L, 300.0 U / L, 500.0 U / L, 1000.0 U / L, 1500.0 U / L, 2000.0 U / L, 2500.0 U / L, and 3000.0 U / L, respectively. Take an equal volume of the functionalized two-zone detection hydrogel prepared in Examples 1-3, immerse it in the prepared alkaline phosphatase solutions, irradiate the hydrogel with a UV lamp, and take pictures simultaneously. Set the originally prepared functionalized zone detection hydrogel as a reference sample.

[0047] Results: The functionalized bi-region detection hydrogels prepared in Examples 1-3 emitted blue fluorescence under ultraviolet light. When immersed in alkaline phosphatase solution, the blue fluorescence of the functionalized bi-region detection hydrogels gradually faded with increasing concentration, and finally disappeared.

Claims

1. A functionalized zoned detection hydrogel, characterized in that, The functionalized zone detection hydrogel is divided into zone one and zone two. Zone one is used to detect hydrogen peroxide in reactive oxygen species produced by bacteria, and zone two is used to detect enzymes produced by bacteria. The first region is obtained by freezing and thawing egg white-gold nanoclusters with polyethylene glycol, using egg white-gold nanoclusters as the functional detection center; the egg white-gold nanoclusters are obtained by reducing tetrachloroauric acid with egg white and cellulose nanocrystals as protective ligands. The second region is obtained by freezing and thawing bovine serum albumin-gold nanoclusters with polyethylene glycol, using bovine serum albumin-gold nanoclusters as the functional detection center; the bovine serum albumin-gold nanoclusters are obtained by reducing tetrachloroauric acid with bovine serum albumin and cellulose nanocrystals as protective ligands. The functionalized partitioned detection hydrogel is prepared by the following method: S1. Preparation of egg white-gold nanoclusters: Tetrachloroauric acid solution was added to a cellulose nanocrystal dispersion, followed by egg white. The mixture was reacted at 30-120°C for 3-48 h to obtain egg white-gold nanoclusters. The concentration of the tetrachloroauric acid solution was 5.0-20.0 mmol / L, the mass percentage of the cellulose nanocrystal dispersion was 1-3 wt%, and the corresponding volume ratio of the tetrachloroauric acid solution to the cellulose nanocrystal dispersion was 1:1-2. The volume ratio of the egg white to the cellulose nanocrystal dispersion was also 1:1-2. S2. Preparation of bovine serum albumin-gold nanoclusters: Tetrachloroauric acid solution was added to a cellulose nanocrystal dispersion, followed by the addition of bovine serum albumin. The mixture was reacted at 30–120 °C for 3–48 h to obtain bovine serum albumin-gold nanoclusters. The concentration of the tetrachloroauric acid solution was 0.1–50.0 mmol / L, the mass percentage of the cellulose nanocrystal dispersion was 1–3 wt%, and the corresponding volume ratio of the tetrachloroauric acid solution to the cellulose nanocrystal dispersion was 1:1–3. The volume ratio of the bovine serum albumin to the cellulose nanocrystal dispersion was also 1:1–3. S3. Preparation of functionalized partitioned detection hydrogel: The egg white-gold nanoclusters obtained in step S1 were added to a polyethylene glycol solution with a mass concentration of 1-50 wt% at a volume ratio of 1-5:1 and stirred for 4-48 h to obtain an egg white-gold nanocluster mixture. The bovine serum albumin-gold nanoclusters obtained in step S2 were added to a polyethylene glycol solution with a mass concentration of 1-50 wt% at a volume ratio of 1-5:1 and stirred for 4-48 h to obtain a bovine serum albumin-gold nanocluster mixture. The resulting egg white-gold nanocluster mixture was poured into a first-zone mold, and the resulting bovine serum albumin-gold nanocluster mixture was poured into a second-zone mold and placed in the freezer compartment of a refrigerator for 3-5 cycles of freeze-thaw. The functionalized first-zone detection hydrogel and the functionalized second-zone detection hydrogel were combined together, and the hydrogels of the two zones adhered tightly together due to their own viscosity to obtain a functionalized zone detection hydrogel.

2. The application of the functionalized zone detection hydrogel of claim 1 in the preparation of hydrogels for monitoring the degree of wound infection, wherein one zone of the functionalized zone detection hydrogel is used to detect hydrogen peroxide in reactive oxygen species produced by bacteria, and the detection sensitivity for hydrogen peroxide reaches 10. -5 The detection process involves functionalizing the hydrogel in zone one, where the red fluorescence is rapidly quenched. A colorimetric method is used to compare the intensity of the red fluorescence to indicate the hydrogen peroxide content. Zone two is used to detect enzymes produced by Gram-negative bacteria, with a sensitivity of 0.01–3000 U / L for alkaline phosphatase secreted by Gram-negative bacteria. During detection, the blue fluorescence of the hydrogel in zone two is quenched, and a colorimetric method is used to compare the intensity of the blue fluorescence to indicate the enzyme content.

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