PEDOT-modified DNA hydrogel as well as preparation method and application thereof

Abstract

The invention discloses PEDOT (poly (3, 4-ethylenedioxythiophene)) modified DNA (deoxyribonucleic acid) hydrogel, which is hydrogel with a three-dimensional network structure, which is formed by (1) PEDOT: a DNA compound and a cross-linking agent, or (2) PEDOT: a DNA compound, long-chain DNA and a cross-linking agent through cross-linking, the PEDOT: DNA compound is a compound which is formed by assembling poly (3, 4-ethylenedioxythiophene) and double-stranded DNA through electrostatic interaction; one chain of the double-chain DNA contains a recognition sequence of the biomarker, and the other chain of the double-chain DNA contains a sequence which is complementary with the recognition sequence of the biomarker and is partially mismatched with the recognition sequence of the biomarker. The invention also discloses an application of the PEDOT modified DNA hydrogel in preparation or as a capacitance material for monitoring a diabetes wound microenvironment. The PEDOT modified DNA hydrogel disclosed by the invention has good biocompatibility, wound protectiveness and capacitance responsiveness, can realize conversion from a biological signal to a capacitance signal, and has great application potential in the aspect of monitoring a diabetes wound microenvironment.

Description

Technical Field

[0001] This invention relates to a PEDOT-modified DNA hydrogel, its preparation method and application, belonging to the field of biosensor materials technology. Background Technology

[0002] Diabetes is a common metabolic disease with an increasing incidence rate worldwide. Diabetic patients have a persistently high blood sugar level due to impaired insulin secretion or action. Long-term hyperglycemia can lead to many complications, such as diabetic wounds and diabetic nephropathy. Among them, diabetic wounds are chronic wounds that are difficult to heal and may eventually lead to amputation or death. Monitoring and management of diabetic wounds are considered as important as treatment in clinical practice. The typical pathological features of diabetic wounds are abnormal acidity and alkalinity, inflammatory outbreaks and bacterial infections, which intertwine to form a vicious cycle: (1) Diabetic wounds are initially acidic (pH about 4.0 to 7.0). However, with long-term exposure to weakly alkaline tissues and interstitial fluid, the wound site becomes persistently alkaline (pH > 7.0). (2) Cells release damage-associated molecular patterns (DAMPs) under injury or stress conditions, activating the immune system and triggering an inflammatory response, especially in macrophages, neutrophils and other cells that secrete pro-inflammatory factors such as TNF-α. In addition, a high-glucose environment can exacerbate the inflammatory response. (3) The alkaline environment of a wound is conducive to bacterial colonization, and bacterial infection can further shift the alkalinity of diabetic wounds and exacerbate the inflammatory response. Therefore, specific dynamic monitoring of wound pH, inflammatory factors, and microbial load can accurately interpret the pathological development process and guide timely adjustment of clinical treatment strategies, providing an important basis for breaking the vicious cycle of "high pH-inflammation-infection". In this case, the development of strategies for wound monitoring is particularly important. Among them, the more common techniques include: using glass electrode pH meters or pH test strips; using ELISA technology to detect specific biomarkers in wound secretions, such as cytokines and enzymes, to determine the inflammatory status of the wound; and using bacterial culture or microscopic examination to assess the degree of wound infection. However, pH meters require large sample volumes, and pH test strips have low detection accuracy; ELISA, bacterial culture, and microscopic observation are time-consuming, cumbersome to operate, and require professional operation, which patients cannot complete on their own for daily monitoring.

[0003] In diabetic wound monitoring, while flexible electrodes directly contacting the wound can provide real-time data support, the physical contact between the flexible electrode and the wound may damage fragile newly formed tissue, disrupt the local wound microenvironment, and thus exacerbate wound deterioration and interfere with tissue repair processes. Furthermore, flexible electrodes have poor adsorption capacity for wound exudate, resulting in low utilization of the exudate. To address these challenges, there is an urgent need to develop novel sensing materials and combine them with flexible electrodes to work together on the wound. When the sensing material identifies biosignals in the wound, it converts them into detectable electrical signals, thereby avoiding the series of harms caused by direct contact between the flexible electrode and the skin or wound. Hydrogels are a material that meets the above requirements, and their porous structure can also efficiently absorb wound exudate. However, the traditional polymeric hydrogel framework itself cannot specifically respond to disease biomarkers, limiting its application potential in diabetic wound monitoring.

[0004] Poly(3,4-ethylenedioxythiophene) (PEDOT) is a conductive polymer material commonly used in biosensors, neuromodulation, and other fields. Currently, there are no reports of using PEDOT combined with DNA for diabetes wound monitoring. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides a PEDOT-modified DNA hydrogel with conductivity and molecular recognition function.

[0006] This invention is achieved through the following technical solution: A PEDOT-modified DNA hydrogel is a hydrogel with a three-dimensional network structure formed by cross-linking ① a PEDOT:DNA complex (pDNA) and a cross-linking agent, or ② a PEDOT:DNA complex, long-chain DNA and a cross-linking agent. The PEDOT:DNA complex is a complex assembled from positively charged poly(3,4-ethylenedioxythiophene) and negatively charged double-stranded DNA through electrostatic interactions. One strand of the double-stranded DNA contains a biomarker recognition sequence (recognition strand), and the other strand contains a partially mismatched sequence complementary to the biomarker recognition sequence (complementary strand). During cross-linking, the primary amine groups on the unpaired bases of the pDNA (and the primary amine groups on the unpaired bases of the long-chain DNA) undergo nucleophilic ring-opening addition reactions with the epoxy groups of the cross-linking agent, forming a three-dimensional network structure.

[0007] Further, the biomarker is selected from any one or more of hydrogen ions, inflammatory factors, and bacteria. Even further, the inflammatory factor is selected from α-tumor necrosis factor (TNF-α); the bacteria are selected from Staphylococcus aureus (…). Staphylococcus aureus, S.aureus ).

[0008] Furthermore, the complementary and partially mismatched structure employs an intermittent mismatch design strategy, specifically: except for the non-complementary polyA segment, one non-complementary base is placed after every 4 to 6 complementary bases to reduce the binding strength between complementary strands, making it lower than the specific interaction between the recognition sequence and the target biomarker, with each strand containing 3 unpaired bases.

[0009] Furthermore, when the biomarker is a hydrogen ion, the corresponding recognition sequence (referred to as the hydrogen ion-responsive sequence) is denoted as DNA. H Its nucleotide sequence is shown in SEQ ID NO:1; when the biomarker is TNF-α, the corresponding recognition sequence (referred to as the TNF-α response sequence) is denoted as DNA. TNF-α Its nucleotide sequence is shown in SEQ ID NO:2; when the biomarker is Staphylococcus aureus, the corresponding recognition sequence (referred to as...) S.aureus Response sequences (denoted as DNA) S.aureus Its nucleotide sequence is shown in SEQ ID NO:3.

[0010] Furthermore, the DNA H The complementary sequence is denoted as cDNA. H Its nucleotide sequence is shown in SEQ ID NO:4; the DNA TNF-α The complementary sequence is denoted as cDNA. TNF-α Its nucleotide sequence is shown in SEQ ID NO:5; the DNA S.aureus The complementary sequence is denoted as cDNA. S.aureus Its nucleotide sequence is shown in SEQ ID NO:6.

[0011] Furthermore, the long-chain DNA is genomic DNA derived from fish or mammals, or chemically synthesized long-chain DNA. Even further, it is salmon sperm DNA, a commercially available product readily available in the market.

[0012] Furthermore, the crosslinking agent is selected from polyethylene glycol diglycidyl ether (PEGDE).

[0013] Further, the mass ratio of pDNA to cross-linking agent is 1:(2-3), preferably 1:2.5; or the mass ratio of pDNA, long-chain DNA, and cross-linking agent is 1:(8-12):(2-3), preferably 1:10:2.5.

[0014] The method for preparing the PEDOT-modified DNA hydrogel includes the following steps: (1) Preparation of PEDOT:DNA complex: In the presence of solvent (anhydrous ethanol) and catalyst ferric p-toluenesulfonate (FepTS), 3,4-ethylenedioxythiophene monomer (EDOT) and double-stranded DNA are assembled to form PEDOT:DNA complex through electrostatic interaction. (2) Preparation of hydrogel: PEDOT:DNA complex, cross-linking agent and catalyst tetramethylethylenediamine (TMEDA) are added to water, or: PEDOT:DNA complex, long-chain DNA, cross-linking agent and catalyst tetramethylethylenediamine are added to water and cross-linked at room temperature and in the dark to form hydrogel.

[0015] Further, the mass ratio of the pDNA, cross-linking agent, and tetramethylethylenediamine is 1:(2-3):(0.4-0.6), preferably 1:2.5:0.5; or the mass ratio of the pDNA, long-chain DNA, cross-linking agent, and tetramethylethylenediamine is 1:(8-12):(2-3):(0.4-0.6), preferably 1:10:2.5:0.5.

[0016] The application principle of the PEDOT-modified DNA hydrogel in the preparation or as a capacitor material for monitoring the microenvironment of diabetic wounds is as follows: (1) When biomarkers are absent or at extremely low concentrations, the pDNA in the hydrogel is always in a double-stranded structure (the molecular circuit is in a "connected" state). At this time, the pDNA molecular conductive network is unobstructed, the carrier migration ability is strong, and the dielectric constant and capacitance of the hydrogel are both in a high state; (2) When biomarkers are present or at high concentrations, the double strands of pDNA in the hydrogel are opened (the molecular circuit is in a "disconnected" state), the conductive network is broken, the carrier migration ability decreases, and the dielectric constant and capacitance of the hydrogel decrease. At this time, the capacitance data is reflected to the capacitance monitoring device through the flexible electrode sheet (realizing the conversion of biological signals to capacitance signals). The capacitance monitoring device then transmits the data to the display device (such as a computer, smartphone, etc.), thereby realizing the monitoring of the pH, inflammation status and infection status of the wound microenvironment. By outputting capacitance signals, it specifically responds to different pathological signals (biomarkers) to monitor the spatiotemporal changes of the microenvironment of diabetic wounds and the pH, inflammation and infection status of diabetic wounds at different stages.

[0017] Furthermore, in specific applications, PEDOT-modified DNA hydrogel is combined with flexible electrode sheets and attached to the wound (the action time can be set to 60 seconds). The capacitance data is transmitted to a smartphone via a portable capacitance detection device (the transmission method can be Bluetooth). The analysis is then used to monitor the pH, inflammation status, and infection status of the wound microenvironment.

[0018] A microenvironment monitoring device for diabetic wounds includes a PEDOT-modified DNA hydrogel, a flexible electrode sheet, a portable capacitance monitoring device, and a smartphone. The PEDOT-modified DNA hydrogel is combined with the flexible electrode sheet, and the portable capacitance monitoring device monitors the capacitance of the flexible electrode sheet and transmits the capacitance data to the smartphone (the transmission method may be Bluetooth).

[0019] The PEDOT-modified DNA hydrogel of this invention exhibits excellent biocompatibility, wound protection, and capacitive responsiveness, enabling the conversion of biological signals into capacitive signals. It shows great potential for application in monitoring the microenvironment of diabetic wounds. Specifically, it has the following beneficial effects: (1) The DNA response sequence and its complementary sequence used in this invention achieve precise control of interstrand affinity by introducing a spaced mismatch design. This strategy effectively avoids interference caused by nonspecific binding and significantly improves the system's sensitivity and selectivity for identifying target biomarkers.

[0020] (2) The pDNA hydrogel of the present invention is a pDNA hydrogel network prepared by the interaction of positively charged PEDOT chains and negatively charged DNA chains to form a PEDOT:DNA complex, and then by the nucleophilic ring-opening addition reaction of the primary amine groups of unpaired bases in pDNA and the primary amine groups of unpaired bases in long-chain DNA with the epoxy groups on PEGDE.

[0021] (3) This invention modifies DNA using PEDOT, giving it capacitive responsiveness. When the hydrogel material is exposed to specific stimuli (such as acidity, alkalinity, inflammatory factors, or bacteria) in a diabetic wound environment, the functional nucleic acid sequence recognizes the target and triggers DNA double-strand dissociation. This conformational change causes the pDNA molecular circuitry to switch from an "connected" state to an "disconnected" state, resulting in the breakage of the pDNA conductive network, a decrease in carrier mobility, and a corresponding decrease in the dielectric constant and capacitance of the hydrogel. This change can be converted into a stable capacitance signal output through an integrated flexible electrode and a portable capacitance detection system. Combined with a wireless Bluetooth module, the data can be transmitted to a mobile terminal in real time for intelligent analysis. This achieves the conversion of biological signals into capacitance signals.

[0022] (4) This invention combines a portable capacitance monitoring device and a flexible electrode sheet to achieve efficient monitoring of the spatiotemporal changes of the microenvironment of diabetic wounds and the pH, inflammation and infection status of diabetic wounds at different stages.

[0023] (5) The pDNA hydrogel of the present invention has excellent flexibility, good biocompatibility, and wound protection, which can minimize the side effects such as wound deterioration caused by direct contact of electrodes with wounds. It can realize continuous monitoring of the microenvironment of diabetic wounds without invasive operation and is suitable for chronic wound management, drug intervention effect evaluation and construction of intelligent wearable medical devices.

[0024] (6) The pDNA hydrogel preparation of the present invention uses electrostatic assembly combined with nucleophilic ring-opening addition reaction to construct hydrogel network. The reaction conditions are mild and do not require high temperature or toxic organic solvents. It has good biocompatibility and potential for large-scale production.

[0025] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0026] Figure 1 : Relative fluorescence intensity changes before and after biomarker recognition by responsive sequences (n=3), where A: hydrogen ion responsive sequence; B: TNF-α responsive sequence; C: S.aureus Response sequence.

[0027] Figure 2 pDNA H Photographs of hydrogels.

[0028] Figure 3 Rheological characterization of pDNA hydrogels, where, from left to right, pDNA... H pDNA TNF-α pDNA S.aureus .

[0029] Figure 4 SEM images of DNA hydrogels and pDNA hydrogels, where, from top to bottom and left to right, the DNA hydrogel is shown. H hydrogel, pDNA H hydrogels, DNA TNF-α hydrogel, pDNA TNF-α hydrogels, DNA S.aureus hydrogel, pDNA S.aureus Hydrogel.

[0030] Figure 5 DNA R hydrogel, pDNA R The results of the dielectric constant and capacitance measurements of the hydrogel, where A: dielectric constant; B: capacitance.

[0031] Figure 6 pDNA H hydrogel, pDNA TNF-α hydrogel, pDNAS.aureus The dielectric constant and capacitance of the hydrogel were measured, where A, B, and C are pDNA, respectively. H hydrogel, pDNA TNF-α hydrogel, pDNA S.aureus Frequency distribution of dielectric constant of hydrogel; D, E, F are pDNA, respectively. H hydrogel, pDNA TNF-α hydrogel, pDNA S.aureus Capacitance diagram of hydrogel.

[0032] Figure 7 pDNA H Photograph of the hydrogel assembled with the flexible electrode sheet.

[0033] Figure 8 Results of specific response validation of pDNA hydrogel (n=3), where A: response to hydrochloric acid (pH=4); B: response to TNF-α (30 pM); C: response to... S.aureus (1×10) 9 The response of CFU / mL; D: pDNA H The response of the hydrogel to different concentrations of hydrochloric acid; E: pDNA TNF-α The response of hydrogels to different concentrations of TNF-α; F: pDNA S.aureus Hydrogels at different concentrations S.aureus The response.

[0034] Figure 9 Linear fitting plot of pDNA hydrogel, where A: pDNA H Linear fitting plot of hydrogel capacitance changes at different pH values; B: pDNA TNF-α Linear fitting plot of hydrogel capacitance changes at different TNF-α concentrations; C: pDNA S.aureus Linear fitting graph of the logarithmic capacitance change of hydrogels with different concentrations of Staphylococcus aureus. The top left corner shows the linear fitting formula and variance.

[0035] Figure 10 : Dynamic monitoring results of the wound microenvironment in untreated diabetic mice, where A: pDNA H Analysis of capacitance changes over time and pH changes after hydrogel adhesion to wound sites in uninfected diabetic mice (n=5); B: pDNA TNF-α Analysis of changes in capacitance over time and TNF-α concentration after hydrogel adhesion to wound sites in uninfected diabetic mice (n=5); C: pDNA at different time points S.aureusAnalysis of changes in capacitance and bacterial concentration over time at the wound site of infected diabetic mice with hydrogel (n=5); D: Bacterial colony smear at the wound site of mice in the wound infection group at different time points; E: Line graph of bacterial colony count at the wound site of mice in the wound infection group at different time points (n=3).

[0036] Figure 11 : Dynamic monitoring results of the wound microenvironment in treated diabetic mice, where A: pDNA H Analysis of capacitance changes over time and pH changes after hydrogel adhesion to wound sites in uninfected diabetic mice (n=5); B: pDNA TNF-α Analysis of changes in capacitance over time and TNF-α concentration after hydrogel adhesion to wound sites in uninfected diabetic mice (n=5); C: pDNA at different time points S.aureus Analysis of changes in capacitance and bacterial concentration over time at the wound site of infected diabetic mice with hydrogel (n=5); D: Bacterial colony smear at the wound site of mice in the wound infection group at different time points; E: Line graph of bacterial colony count at the wound site of mice in the wound infection group at different time points (n=3).

[0037] Figure 12 RT-qPCR was used to analyze the relative mRNA expression level of TNF-α in wound tissue, where A: treatment group; B: non-treatment group. Detailed Implementation

[0038] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0039] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0040] Experiment 1: Validation of Biomarkers for Responsive Nucleic Acid Sequences (1) To interpret pathological issues such as abnormal acid-base balance, inflammatory outbreaks, and bacterial infections in diabetic wounds, this study used hydrogen ions, TNF-α, S.aureus As biomarkers, hydrogen ions, TNF-α, and S.aureus The responsive DNA sequences are named DNA. H DNA TNF-α and DNA S.aureus And designed a random sequence DNA R(As a control). Then, complementary sequences were designed based on the base pairing principle. To regulate inter-strand affinity, an intermittent mismatch strategy was adopted: after removing the non-complementary polyA region, one non-complementary base was inserted for every five complementary bases to reduce the binding strength between complementary strands, making it lower than the specific interaction between nucleic acids and target biomarkers. Each strand contained three unpaired bases. The designed complementary sequences were named cDNA. R cDNA H cDNA TNF-α and cDNA S.aureus .

[0041] DNA H The nucleotide sequence is shown in SEQ ID NO:1, as shown below (direction 5'-3'): AAAAAAAAAAAAAAAAAAAAAAAAAAACCCTAACCCTAACCCTAACCCTA.

[0042] DNA TNF-α The nucleotide sequence is shown in SEQ ID NO:2, as shown below (direction 5'-3'): AAAAAAAAAAAAAAAAAAAAAAAAAAAGCGCCACTACAGGGGAGCTGCCATTCGAATAGGTGGGCCGC.

[0043] DNA S.aureus The nucleotide sequence is shown in SEQ ID NO:3, as shown below (direction 5'-3'): AAAAAAAAAAAAAAAAAAAAAAAAAAATCGGCACGTCAGTAGCGCTCGCTGGTCATCCCACAGCTACGTC.

[0044] cDNA H The nucleotide sequence is shown in SEQ ID NO:4, as shown below (direction 5'-3'): AAAAAAAAAAAAAAAAAAAAAAAAAAATAGTGTTAGTGTTAGTGTTAGGG.

[0045] cDNA TNF-α The nucleotide sequence is shown in SEQ ID NO:5, as shown below (direction 5'-3'): AAAAAAAAAAAAAAAAAAAAAAAAGCGGCCAACCTAGTCGAACGGCAG.

[0046] cDNA S.aureus The nucleotide sequence is shown in SEQ ID NO:6, as shown below (direction 5'-3'): AAAAAAAAAAAAAAAAAAAAAAAAAAAGACGTACCTGTGGTATGAACAGCG.

[0047] DNA R The nucleotide sequence is shown in SEQ ID NO:7, as shown below (direction 5'-3'): AAAAAAAAAAAAAAAAAAAAAAAAAAACGTCGATCGATCGAT.

[0048] cDNA R The nucleotide sequence is shown in SEQ ID NO:8, as shown below (direction 5'-3'): AAAAAAAAAAAAAAAAAAAAAAAAAAATCGATCGATCGACG.

[0049] (2) Preparation of nucleic acid aptamers (recognition strands) and complementary strands: The above DNA molecules were prepared using a DNA synthesizer.

[0050] (3) Investigate hydrogen ion-responsive DNA sequences using the SYBR Green I fluorescent probe. H (and its complementary sequence cDNA) H Double-stranded DNA H / cDNA H In the conformational dynamics of the hydrogen ion response, the results are as follows: Figure 1 As shown in Figure A. The results show that double-stranded DNA H / cDNA H The fluorescence intensity of the double-stranded DNA was significantly higher than that of the single-stranded DNA control group (pH 7.0), which is due to the specific enhancement of SYBR Green I fluorescence intensity by the double-stranded structure. In an acidic environment (pH 5.0), the fluorescence intensity decreased to 76.1% of the initial value, indicating that a large number of double-stranded DNA structures responded to hydrogen ions and became single-stranded structures. This contrasts with DNA sequences that respond to TNF-α. TNF-α (and its complementary cDNA sequence) TNF-α Double-stranded DNA TNF-α / cDNA TNF-α ), S.aureus Response sequence DNA S.aureus (and its complementary cDNA sequence) S.aureus Double-stranded DNA S.aureus / cDNA S.aureusThe expected decrease in fluorescence was not observed in SYBR Green I fluorescence detection, suggesting that the high concentration of target protein and the double-stranded DNA in the bacteria affected the experiment.

[0051] Therefore, this study designed a molecular fluorescent probe, namely, a probe for DNA fluorescence. TNF-α and DNA S.aureus The 3' end of the cDNA is modified with 5-FAM fluorescence, and its complementary sequence is then used. TNF-α and cDNA S.aureus The 5' end of the FAM is labeled with a BHQ-1 quencher group. When the double strands are in a complementary state, the distance between the FAM (donor) and BHQ-1 (acceptor) is <10 nm, satisfying the FRET resonance condition. At this time, the emission spectrum of the FAM (λ) is... em =520 nm) and the absorption spectrum of BHQ-1 (λ max Effective overlap occurs at 535 nm, resulting in a significant decrease in fluorescence signal. When the target (TNF-α or...) is effectively overlapped, the fluorescence signal is significantly reduced. S.aureus ) and DNA TNF-α and DNA S.aureus After binding to their respective functional DNA sequences, the double-stranded structure dissociates, increasing the distance between the FAM at the end of the functional sequence and the BHQ-1 at the end of the complementary sequence, allowing the FAM group to be re-excited. Based on this, the double-stranded DNA can be detected... TNF-α / cDNA TNF-α and DNA S.aureus / cDNA S.aureus The changes in FAM fluorescence signal were used to verify the responsive structural changes of the designed inflammatory factor-responsive sequences and Staphylococcus aureus-responsive sequences in the presence of corresponding biomarkers. The results are as follows: Figure 1 As shown in B and C. The experimental results show that DNA TNF-α / cDNA TNF-α Groups and DNA S. aureus / cDNA S.aureus The fluorescence intensity of the group was significantly lower than that of the corresponding control group. This is because the formation of DNA double helix leads to the FRET effect between the FAM group and the BHQ-1 group, resulting in the quenching of the FAM group, indicating the formation of double-stranded molecules. In TNF-α and S.aureus In the environment in which DNA exists TNF-α / cDNA TNF-α Groups and DNA S.aureus / cDNA S.aureus The fluorescence intensity of the two groups increased significantly to 2.9 times and 3.2 times the original, respectively, indicating that a large number of double-stranded DNA structures were transformed into single-stranded structures in response to inflammatory factors and bacteria.

[0052] Therefore, the DNA sequence designed above (DNA) H DNA TNF-αDNA S.aureus It can specifically identify corresponding biomarkers (H) + TNF-α S.aureus The molecular switch function was verified by triggering fluorescence signal output through conformational changes. This provides theoretical support for further verification of the responsiveness of DNA sequences in hydrogel networks and evaluation of their target recognition ability and therapeutic effects in in vivo models.

[0053] Example 1: Preparation of PEDOT-modified DNA hydrogel The steps are as follows: (1) Preparation of pDNA DNA H With cDNA H The powder was dissolved in water to prepare a 1000 nM solution; 100 μL of each solution was placed in an EP tube, allowed to stand for 12 hours, concentrated under vacuum and dried. Add 1 mL of anhydrous ethanol, 0.2847 mg of FepTS and 0.0711 mg of EDOT to an EP tube and keep it at 4°C for 30 min. The positively charged PEDOT and the negatively charged double-stranded DNA will assemble into a PEDOT:DNA complex (pDNA) through electrostatic interaction. After the reaction was complete, the supernatant was discarded, and the solid product was washed three times with 1 mL of anhydrous ethanol and dried at room temperature for 30 minutes to obtain pDNA. H Similarly, pDNA was prepared. TNF-α pDNA S.aureus pDNA R .

[0054] (2) Preparation of pDNA hydrogel The pDNA (1 mg) prepared above was dissolved in 86 mg of water along with 10 mg of salmon sperm DNA. Then, 2.5 mg of cross-linking agent PEGDE and 0.5 mg of catalyst TMEDA were added and mixed thoroughly. The solution was poured into a mold (7 mm radius, 1 mm thickness) and cross-linked for two days at room temperature in the dark. (The primary amine groups on the unpaired bases of both pDNA and salmon sperm DNA undergo nucleophilic ring-opening addition reactions with the epoxy groups of the cross-linking agent, forming a three-dimensional network structure.) This resulted in a pDNA hydrogel. The randomly non-responsive hydrogels prepared were denoted as pDNA hydrogels. R Hydrogels, hydrogen ion-responsive hydrogels are denoted as pDNA. H Hydrogels (such as) Figure 2 As shown in the figure, the inflammatory factor-responsive hydrogel is denoted as pDNA. TNF-α Hydrogels, Staphylococcus aureus responsive hydrogels are denoted as pDNA.S.aureus Hydrogel.

[0055] Experiment 2 Mechanical properties and structural characterization of pDNA hydrogels The pDNA hydrogel prepared in Example 2 above was tested for mechanical properties using an Anton Paar rheometer, and its surface morphology was observed using SEM. The specific process is as follows: (1) Rheological properties pDNA H / pDNA TNF-α / pDNA S.aureus Hydrogel (100 μL) was mounted on a 20 mm parallel plate. The test frequency varied from 0.1 to 100 Hz, and all measurements were performed at room temperature under a constant stress of 100 Pa.

[0056] The results are as follows Figure 3 As shown in the figure, this experiment yielded a mechanically stable hydrogel. Furthermore, after modification with PEDOT, the overall mechanical strength of the hydrogel was improved to a certain extent, indicating that PEDOT was successfully modified onto the DNA.

[0057] (2) Morphological structure characterization Different DNA gels (where DNA) were used to prepare different DNA gels. H hydrogels, DNA TNF-α hydrogels, DNA S.aureus The preparation method of the hydrogel is the same as in Example 1, except that after obtaining the double-stranded DNA, it is not reacted with EDOT, but directly proceeds to step 2). After lyophilization, the surface morphology of the gel is observed by SEM, and the results are as follows. Figure 4 As shown, the hydrogel exhibits a microporous structure similar to typical hydrogels. This three-dimensional network structure facilitates the diffusion of macromolecules, reducing the hydrogel's response time and increasing the sensitivity of DNA within the hydrogel to various biomarkers. Furthermore, the hydrogel structure, when applied to a wound, does not impede wound ventilation, thus preventing suppuration or the growth of anaerobic bacteria. Simultaneously, it is evident that PEDOT modification of DNA does not affect the three-dimensional network structure of the hydrogel.

[0058] Experiment 3: Changes in capacitance and dielectric constant of pDNA hydrogel To verify the feasibility of using hydrogels to detect the capacitive response of biomarkers, this study first determined the dielectric constants of DNA hydrogels and pDNA hydrogels. The coaxial method was used: the hydrogel dimensions were 3.06 mm inner diameter, 6.97 mm outer diameter, and 1 mm thickness, with an accuracy requirement of ±0.02 mm, and the testing frequency ranged from 1 to 18 GHz.

[0059] Data processing method: For data where the capacitance changes, take the average of 5 data points of the smoothed line segment before the change as C0, and take the average of 5 data points of the smoothed line segment after the change as C1; for data where the capacitance does not change, take the average of 5 data points of the smoothed line segment within the first 5 seconds as C0, and take the average of 5 data points of the smoothed line segment in the last 5 seconds as C1, and obtain ΔC=(C1-C0), and then calculate the required data ΔC / C0.

[0060] DNA R hydrogel, pDNA R The results of the hydrogel assay are as follows: Figure 5 As shown in the figure. Experimental results show that the average dielectric constant of the DNA hydrogel increased significantly by about 2.26 times and the capacitance increased by about 1.84 times after PEDOT doping.

[0061] Furthermore, regarding pDNA H Hydrogel (hydrochloric acid-free) and pDNA H Hydrogel (hydrochloric acid, pH=4), pDNA TNF-α Hydrogel (TNF-α-free) and pDNA TNF-α Hydrogel (30 pM TNF-α), pDNA S.aureus Hydrogel (None) S.aureus ) and pDNA S.aureus Hydrogel (1×10) 9 CFU / mL S.aureus The capacitance change was detected using a portable capacitance analyzer; the dielectric constant was determined using a microwave network vector analyzer / vector network analyzer. Results are as follows: Figure 6 As shown, the capacitance and dielectric constant of each pDNA hydrogel decreased to varying degrees. This phenomenon may be related to the differences in the sensitivity of nucleic acid sequences to biomarkers.

[0062] Experiment 4: In vitro specific capacitance response of pDNA hydrogel This experiment verifies the feasibility of using the pDNA hydrogel of this invention for specific recognition of biomarkers through in vitro experiments.

[0063] A Bluetooth-enabled smartphone was connected to the capacitance monitoring box; flexible electrode sheets were assembled with different groups of hydrogels, and the assembled images are shown below. Figure 7 As shown.

[0064] Verify the specificity of the hydrogel: Add hydrochloric acid (pH=4) and TNF-α (30 pM) respectively. S.aureus (1×10) 9 Add 1-2 μL of CFU / mL and pure water to the DNA. R DNA H pDNAH DNA TNF-α pDNA TNF-α DNA S.aureus pDNA S.aureus On the hydrogel surface, the capacitance value on the device was read for 60 seconds, and the data was exported. Three parallel experiments were performed for each sample.

[0065] For pDNA H pDNA TNF-α pDNA S.aureus Further experiments were conducted using hydrogels: different concentration gradients of hydrochloric acid (pH=5, 5.5, 6, 6.5, 7) and TNF-α (0.3, 0.5, 0.75, 1, 1.25, 1.5 pM) were used. S.aureus (1×10 3 1×10 5 1×10 7 1×10 9 1×10 11 1×10 13 1–2 μL of each of the CFU / mL biomarkers was dropped onto the corresponding hydrogel surface and left for 60 s. Data were then exported, and a linear fit was performed on the relationship between the concentration of different biomarkers and the change in capacitance. Three parallel experiments were conducted for each sample.

[0066] The results are as follows Figure 8 As shown, the pDNA hydrogel only responded to the corresponding biomarker, while others did not, a response reflected in the decrease in capacitance. The unmodified DNA hydrogel, however, did not respond, indicating that PEDOT modification of the responsive nucleic acid sequence effectively improved the capacitance responsiveness of the DNA strand and even the original DNA hydrogel system, greatly aiding in the visualization of biological signals. Furthermore, it can be seen that for biomarkers with concentration gradients, the decrease in capacitance follows a decreasing trend with decreasing concentration, which is consistent with expectations. A linear fit was performed between the biomarker concentration and the change in capacitance, yielding a series of relationships (R0). 2 >0.99), such as Figure 9 As shown, based on this, the corresponding biomarker concentration can be derived after obtaining the change in capacitance, thus enabling quantitative analysis of biomarkers in location samples or wound exudate.

[0067] Experiment 5 Animal Experiment This experiment, conducted on animals, tested the effectiveness of the pDNA hydrogel of this invention in monitoring the wound microenvironment in diabetic mice under both treated and untreated conditions.

[0068] (I) Experimental Procedure A diabetic mouse model was established by intraperitoneal injection of 50 mg / kg streptozotocin daily for one week using several male C57BL / 6 mice aged 2–3 weeks. A 5 mm diameter wound was created on the back of the mouse. The mice were grouped as follows: (1) Non-treatment, non-contaminated group (no treatment was given); (2) Non-treatment infected group (1×10⁻⁶ bacteria were added at 15 min after modeling) 9 Staphylococcus aureus at CFU / mL (with Escherichia coli as a control). (3) Treatment of non-infected groups (dexamethasone acetate was used to treat the wound for inflammation at 3, 24 and 48 h); (4) Treatment of the infected group (1×10⁻⁶ drops were added 15 min after modeling) 9 Staphylococcus aureus at CFU / mL was treated with tetracycline hydrochloride at 3, 24, and 48 h for antibacterial purposes.

[0069] In this experiment, pDNA was used. R Hydrogel monitoring served as a control, while the experimental group used pDNA hydrogels. H hydrogel, pDNA TNF-α hydrogels and pDNA S. aureus Hydrogel.

[0070] After establishing a wound model in mice, pDNA was used. R hydrogel, pDNA H hydrogel, pDNA TNF-α The hydrogel (assembled with flexible electrode pads) was used to monitor the wound microenvironment of mice in the non-bacterial infection group at 0, 3, 6, 9, 24, 48, and 72 h, and pDNA was used. R hydrogel, pDNA S.aureus Hydrogels were used to monitor the microenvironment of the wounds of mice infected with bacteria at 0, 3, 6, 9, 24, 48, and 72 h. Wound tissue from mice at 0, 3, 6, 9, 24, 48, and 72 h after anesthesia was removed and fixed in 4% paraformaldehyde for qRT-PCR analysis. For the bacterial infection group, cotton swabs were moistened with physiological saline and repeatedly applied to the wound surface. The swabs were then immersed in 1 mL of physiological saline in EP tubes, and the bacterial colonies on the wound surface were counted using the plate count method and photographed for comparison.

[0071] (II) Experimental Results (1) Monitoring changes in the microenvironment of untreated diabetic wounds Dynamic monitoring results of the wound microenvironment in untreated diabetic mice, as follows: Figure 10 As shown.

[0072] In the process of examining the natural evolution of wounds in diabetic mice, it was found that pDNA was generated after contact with wound exudate. H The capacitance of the hydrogel showed a decreasing trend from 0 to 9 hours, an increasing trend from 9 to 24 hours, and remained basically unchanged from 24 to 72 hours. Figure 10 A). Subsequently, the pH changes at the wound site were revealed after curve fitting. The results showed that the pH continuously increased over time (0–9 h), indicating that the diabetic wound was in the acute wound phase. After 24 h, the wound pH became alkaline, exhibiting typical characteristics of a chronic wound. Since the experimental animals were housed in an SPF-grade environment, bacterial infection-induced pH increases were ruled out. Therefore, the pH increase was likely due to chronic inflammation and interstitial fluid inflow into the unhealed wound in diabetic patients. Since a persistently alkaline environment has been proven to be closely related to wound deterioration and an increased risk of secondary infection, monitoring changes in the pH of the diabetic wound microenvironment is of great significance for assessing wound progression and preventing secondary diseases. Furthermore, the pH value calculated from the relative change in capacitance showed a high degree of consistency with the value measured by pH test strips, but its accuracy was higher than that of pH test strips. Figure 10 A). Simultaneously, pDNA TNF-α After contact with wound exudate, the relative capacitance of the hydrogel showed a continuous decreasing trend from 0 to 72 hours, indicating that the inflammation level at the wound site gradually intensified over time. Curve fitting analysis revealed that the TNF-α concentration in the wound exudate showed a continuous increasing trend throughout the monitoring period, reaching a peak of 2.3 pM. Figure 10 B), consistent with the trend of RT-qPCR analysis results of wound skin tissue ( Figure 12 A). This indicates that the inflammation of the wound has worsened, leading to poor wound healing, a situation consistent with reports in other literature.

[0073] In addition, continuous monitoring of bacterial proliferation after diabetic wound infection was conducted. Figure 10 C). pDNA at the site of E. coli infection. S.aureus The hydrogel did not show a significant response to *E. coli*. Conversely, at the site of a wound infected with *Staphylococcus aureus*, pDNA... S.aureus The continuous decrease in hydrogel capacitance indicates that the number of Staphylococcus aureus at the wound site gradually increased over time. Fitting the obtained Staphylococcus aureus bacterial count revealed that the colony count in the 0–72 h infection group showed an approximately exponential growth, a result largely consistent with the bacterial count growth trend obtained by the bacterial plating method. Figure 10 D, E).

[0074] (2) Monitoring changes in the microenvironment of diabetic wounds during treatment Dynamic monitoring results of the wound microenvironment in treated diabetic mice are as follows: Figure 11 As shown.

[0075] This study further evaluated the use of pDNA hydrogel capacitive sensing materials to monitor changes in the microenvironment of diabetic wounds during treatment with anti-inflammatory drugs (dexamethasone acetate) and antibacterial drugs (tetracycline hydrochloride). Multiparameter monitoring data were compared between the treatment and non-treatment groups to assess the monitoring capability of pDNA hydrogel in diabetic wound treatment. The results showed that pDNA… H After the hydrogel came into contact with wound exudate, its capacitance showed a decreasing trend from 0 to 9 hours, and slowly increased from 9 to 72 hours. Figure 11 A). Subsequently, the pH value at the wound site was obtained by converting the relative change in capacitance using a fitted curve. The results showed that the wound microenvironment of uninfected mice treated with dexamethasone exhibited acidic homeostasis characteristics within 3–72 h (7.0→5.2→5.6), which was highly consistent with the pH test strip results. Notably, the pH value of the treatment group remained consistently acidic (<6.0), significantly different from the alkalinization trend in the non-treatment group. Figure 10 A). This acidic environment has been proven to effectively inhibit pathogenic bacterial colonization. Simultaneously, pDNA... TNF-α The relative capacitance of the hydrogel showed different changes after contact with wound exudate. Calculations using fitted curves revealed that the TNF-α concentration in the wound exudate did not significantly increase throughout the monitoring period, remaining at a low level (approximately 0.6–0.8 pM), close to the normal physiological inflammation level (0.027–0.415 pM). Figure 10 B). This change pattern is highly consistent with the resolution process of the inflammatory phase in normal wound healing (12–24 h). RT-qPCR results (e.g.) Figure 12 (As shown) this trend was further validated, showing that TNF-α expression levels were synchronously downregulated ( Figure 12 B) indicates that the local inflammatory response has been effectively controlled and the tissue repair process has begun. Furthermore, continuous monitoring of bacterial proliferation in diabetic wounds following Staphylococcus aureus infection and antibiotic treatment was conducted. Figure 11 C). pDNA S.aureus Hydrogel monitoring revealed a rapid decrease in Staphylococcus aureus load after antibacterial treatment, with the colony count decreasing from an initial 1×10⁻⁶. 9 The CFU / mL level dropped sharply to below the threshold for clinical diagnosis of infection (1×10⁻⁶). 5 CFU / mL), and decreased to almost undetectable levels after 72 h ( Figure 11 (D, E). The results are basically consistent with the bacterial count trend obtained by plate count, further demonstrating the high sensitivity and accuracy of hydrogel capacitive sensing materials in dynamic bacterial identification.

[0076] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A PEDOT-modified DNA hydrogel, characterized in that: The PEDOT modified DNA hydrogel is a hydrogel with a three-dimensional network structure crosslinked by ① a PEDOT:DNA complex and a crosslinking agent, or ② a PEDOT:DNA complex, long-chain DNA and a crosslinking agent; the PEDOT:DNA complex is a complex assembled by poly 3,4-ethylenedioxythiophene and double-stranded DNA through electrostatic interaction; one strand of the double-stranded DNA contains a recognition sequence of a biomarker, and the other strand contains a sequence complementary to the recognition sequence of the biomarker and partially mismatched.

2. The PEDOT-modified DNA hydrogel according to claim 1, characterized in that: The biomarker is selected from any one or two or more of hydrogen ions, inflammatory factors and bacteria.

3. The PEDOT-modified DNA hydrogel of claim 2, wherein: The inflammatory factor is selected from TNF-α; and the bacteria are selected from Staphylococcus aureus.

4. The PEDOT-modified DNA hydrogel according to claim 1 or 2 or 3, characterized in that: The biomarker is hydrogen ion, the corresponding recognition sequence is recorded as DNA H , the nucleotide sequence of which is shown as SEQ ID NO: 1; the biomarker is TNF-α, the corresponding recognition sequence is recorded as DNA TNF-α , the nucleotide sequence of which is shown as SEQ ID NO: 2; the biomarker is Staphylococcus aureus, the corresponding recognition sequence is recorded as DNA S.aureus , the nucleotide sequence of which is shown as SEQ ID NO: 3; The DNA H The complement of said DNA is denoted cDNA H The nucleotide sequence of which is represented by SEQ ID NO: 4; the DNA TNF-α The complement of said DNA is denoted cDNA TNF-α The nucleotide sequence of which is represented by SEQ ID NO: 5; the DNA S.aureus The complement of said DNA is denoted cDNA S.aureus The nucleotide sequence of which is represented by SEQ ID NO:

6.

5. The PEDOT-modified DNA hydrogel of claim 1, wherein: The long-chain DNA is genomic DNA of fish or mammalian origin or chemically synthesized long-chain DNA.

6. The PEDOT-modified DNA hydrogel according to claim 1 or 5, characterized in that: The long-chain DNA is selected from salmon sperm DNA; and the crosslinking agent is selected from polyethylene glycol diglycidyl ether.

7. The PEDOT-modified DNA hydrogel of claim 1, wherein: The mass ratio of the pDNA and the crosslinking agent is 1:(2-3); or the mass ratio of the PEDOT:DNA complex, the long-chain DNA and the crosslinking agent is 1:(8-12):(2-3).

8. A method for preparing the PEDOT-modified DNA hydrogel according to any one of claims 1 to 7, characterized in that, The method comprises the following steps: (1) preparing a PEDOT:DNA complex: 3,4-ethylenedioxythiophene monomers and double-stranded DNA are assembled to form a PEDOT:DNA complex through electrostatic interaction under the action of a solvent and a catalyst, iron p-toluenesulfonate; (2) preparing a hydrogel: adding the PEDOT:DNA complex, a crosslinking agent and a catalyst, tetramethyl ethylenediamine, into water, or: adding the PEDOT:DNA complex, long-chain DNA, a crosslinking agent and a catalyst, tetramethyl ethylenediamine, into water, and crosslinking at room temperature under light-proof conditions to form a hydrogel.

9. Use of the PEDOT modified DNA hydrogel of any one of claims 1-7 in the preparation or as a capacitive material for monitoring the microenvironment of a diabetic wound.

10. A diabetic wound microenvironment monitoring device, characterized by: The PEDOT modified DNA hydrogel, a flexible electrode sheet, a portable capacitive monitoring device and a smart phone are combined, the portable capacitive monitoring device monitors the capacitance of the flexible electrode sheet and transmits the capacitive data to the smart phone.

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