A series of controlled release nucleic acid nano probe composite hydrogel and its application in diagnosis and treatment of wound infection

Abstract

This invention belongs to the field of medical technology, specifically relating to a tandem controlled-release nucleic acid nanoprobe composite hydrogel and its application in the diagnosis and treatment of wound infections. First, the invention modifies the surface of gold nanoparticles with nucleic acid sequences via Au-S bonds to prepare a DNA-gold nanoparticle probe (NAFP), achieving highly specific detection of sulfides, a wound infection marker. Then, using mesoporous silica as a carrier, an antibacterial drug is loaded, constructing mesoporous silica D@MSNs loaded with a double-stranded DNA molecule valve-type bactericide. Subsequently, the NAFP and D@MSNs are formulated into a gel to obtain a therapeutic dressing. This dressing can trigger conformational changes in nucleic acids through infection markers, achieving precise matching of drug release to the degree of infection, thereby enabling in-situ detection of bacteria. It also possesses highly efficient antibacterial activity, physical protection, and moisturizing functions. The dressing is easy to operate, has low clinical translation costs, and is suitable for the diagnosis and treatment of acute and chronic wound infections, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a tandem controlled-release nucleic acid nanoprobe composite hydrogel and its application in the diagnosis and treatment of wound infections. Background Technology

[0002] Wound infection is a common clinical complication, especially in chronic wounds such as diabetic foot ulcers and pressure sores. The healing process of these wounds is highly susceptible to bacterial invasion, leading to prolonged and difficult-to-heal infections. In severe cases, it can cause tissue necrosis, sepsis, and other adverse consequences, placing a heavy burden on both the patient's physical and mental health and medical resources. Bacterial infection induces specific changes in the wound microenvironment. By utilizing the conformational response of nucleic acid molecules, precise identification of infection can be achieved, thus providing a core target for the precise diagnosis and treatment of chronic wounds.

[0003] Currently, the diagnosis of wound infections in clinical practice still relies primarily on traditional methods, mainly including clinical symptom observation, bacterial culture, and drug sensitivity testing. Among these, clinical symptom observation is highly dependent on the physician's clinical experience, which is not only subjective but also has low early identification accuracy, easily delaying the optimal intervention time for infection. Although bacterial culture and drug sensitivity testing are the "gold standard" for diagnosis, the testing cycle is long, taking 24-48 hours, and the operation procedure is relatively cumbersome. At the same time, the sensitivity for detecting low-fiber bacterial infections is insufficient, making it difficult to meet the clinical needs for rapid screening in the early stages of infection. At the treatment level, existing protocols are centered on antibiotic application, divided into systemic and topical medications. While systemic intravenous or oral antibiotics can exert broad-spectrum antibacterial effects, their targeting at the wound site is poor, easily leading to side effects such as the proliferation of systemic drug-resistant bacteria, intestinal flora imbalance, and liver and kidney damage. Topical antibiotic preparations (such as ointments and gels) can increase the drug concentration at the wound site, but they suffer from drawbacks such as difficulty in precisely controlling the drug release rate and low bioavailability. Excessive local drug concentration can cause skin irritation, while insufficient concentration can lead to treatment failure, further exacerbating the risk of drug resistance. Existing research on disease biomarker detection and drug release is mostly based on independent systems, lacking a closed-loop control mechanism that directly links infection detection signals and drug release. This results in a severe disconnect between drug release and infection status, leading to problems such as premature release and dosage imbalance. Therefore, developing a tandem controlled-release system integrating "infection detection - signal transduction - on-demand release of anti-inflammatory drugs" is of great significance for improving the accuracy and applicability of wound treatment. Summary of the Invention

[0004] To overcome the shortcomings of the existing technology, this invention designs an integrated dressing for the diagnosis and treatment of wound infection based on a nucleic acid fluorescent nanoprobe (NAFP) and a bactericidal drug-loaded mesoporous silica (D@MSNs). Furthermore, it constructs a tandem regulatory system for infection marker detection, nucleic acid conformation switching, and on-demand release of anti-inflammatory drugs, which can achieve specific in-situ diagnosis of infection foci and nucleic acid-responsive targeted release of bactericidal drugs.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a method for preparing a tandem controlled-release nucleic acid nanoprobe composite hydrogel, the method comprising the following steps: S1. Construction of DNA-gold nanoparticle probe NAFP: S11. Synthesis of gold nanoparticles (AuNPs) by citric acid reduction method: Mix chloroauric acid aqueous solution with water, heat to 70-80℃, add trisodium citrate solution until the solution color turns dark red, continue heating and stirring to remove residual chemical substances in the solution, and finally obtain AuNPs solution. S12. Preparation of DNA-gold nanoparticle probe NAFP: The ribozyme strand Tz shown in SEQ ID No. 2 and the blocking strand Sc shown in SEQ ID No. 3 were mixed, and then silver nitrate solution was added. After incubation, the Tz-Ag-Sc complex was obtained. Then, Tz-Ag-Sc was mixed with the signal probe Fp shown in SEQ ID No. 1 and AuNPs solution. By shaking, Au-S bonds were used to connect Tz-Ag-Sc and Fp to the surface of AuNPs, thus obtaining the final product. S2. Constructing mesoporous silica D@MSNs loaded with bactericides: S21. Preparation of mesoporous silica MSNs: CTAB was dissolved in water, and then NaOH aqueous solution was added. The temperature of the mixture was adjusted to 75-85 ℃, and then tetraethyl orthosilicate (TEOS) was added. The reaction was continued until a white precipitate was formed. The solid crude product was then separated, washed and dried to obtain mesoporous silica MSNs. S22. MSNs are dispersed in anhydrous toluene, and then 3-chloropropyltrimethoxysilane (ClTMS) is added. After the reaction, chloropropyl-modified MSN, i.e., MSN-Cl, is obtained. Then, MSN-Cl is added to a mixed solution of ethanol and concentrated hydrochloric acid, and then refluxed, centrifuged, washed and dried to remove the surfactant CTAB. S23. The purified MSN-Cl was dispersed in a sodium azide-saturated N,N-dimethylformamide (DMF) solution. After stirring at 85-95 °C for 10-15 h, the particles were separated by centrifugation. The particles were then dispersed in PBS buffer, and the residual N,N-dimethylformamide (DMF) solution in the mesopores was removed by stirring. After drying, the azide-treated MSN, i.e., MSN-N3, was obtained. S24. MSN-N3 is dispersed in Tris-HNO3 buffer, antibacterial agent is added, and after incubation, alkyne-functionalized double-stranded DNA as shown in SEQ ID No. 4 and 5 is added. Then CuBr solution is added, and after reaction, residual DNA and physically adsorbed antibacterial agent are removed. Finally, it is dispersed in PBS solution to form D@MSNs with double-stranded DNA as "molecular valve". S3. Preparation of nucleic acid nanoprobe composite hydrogel: Sodium alginate and sodium carboxymethyl cellulose were mixed and dissolved in water to prepare a dressing precursor solution. Glycerin was then added and stirred until the system was homogeneous and transparent. The prepared NAFP probe was mixed with D@MSNs and added to the above precursor solution. The resulting mixture was cross-linked in a mold with calcium chloride solution to obtain the nucleic acid nanoprobe composite hydrogel.

[0006] The nucleic acid nanoprobe composite hydrogel of this invention belongs to a tandem regulation system, comprising a detection module, a treatment module, and a therapeutic system: (1) Detection Module: The nanoprobe in this module uses gold nanoparticles (AuNPs) as the core carrier. Its surface is modified with three nucleic acid molecules via Au-S bonds: a targeted ribozyme chain (Tz), a nucleic acid blocking chain (Sc), and a fluorescently labeled sensing probe (Fp), forming a stable conformation-locked sensing system. The nucleic acid blocking chain contains a complementary sequence to the ribozyme core sequence and a wound infection site H2S release response sequence: the complementary inhibition sequence specifically pairs with the core sequence of the targeted ribozyme chain, blocking its cleavage activity; the response sequence senses changes in hydrogen sulfide in the wound infection microenvironment, causing the C-Ag-C coordination structure to disintegrate. As a specific substrate for the ribozyme chain, the sensing probe contains a cleavage site identifiable by the ribozyme core sequence and is terminally modified with a fluorescent group (FAM). Due to the fluorescence quenching effect of the gold nanoparticles, the FAM fluorescence of the sensing probe is in a closed state.

[0007] When the wound is uninfected, the Sc chain binds tightly to the Tz chain through complementary pairing and the dual action of the C-Ag-C coordination structure, blocking ribozyme cleavage activity. The signal probe is stably attached to the surface of the gold nanoparticles, fluorescence is quenched, and no detection signal is generated. When the wound becomes infected with bacteria, H2S produced by the pathogen's metabolism specifically reacts with Ag+ in the C-Ag-C coordination structure, causing Ag+ to detach from the coordination structure, breaking the coordination effect, and separating the Tz chain from the blocking chain. The Tz chain regains its cleavage activity, and its core sequence accurately identifies and cleaves specific sites of Fp, causing the signal probe to detach from the surface of the gold nanoparticles. The FAM fluorescence signal is restored, and the location and degree of infection can be assessed by the presence or absence and intensity of the fluorescence signal.

[0008] (2) Treatment Module: This module uses mesoporous silica (MSNs) as a drug carrier. Utilizing its uniform pore size and large specific surface area, the bactericide is encapsulated within the pores through physical adsorption. Simultaneously, double-stranded DNA (dsDNA) is loaded onto the surface of the mesoporous silica, acting as a "molecular valve" to regulate drug release. One strand of this double-stranded DNA has a specific complementary relationship with the signal probe sequence detached from the detection module. Through the steric hindrance effect of the double-stranded DNA, the pores of the mesoporous silica are blocked, and the bactericide is stably encapsulated within the pores, preventing premature release.

[0009] When the detection module triggers a fluorescence signal, the detached signal probe specifically pairs and binds to the complementary strand of double-stranded DNA on the surface of mesoporous silica (MSNs), causing the double-stranded DNA to unwind into single-stranded DNA. This eliminates steric hindrance, opens the pores of the mesoporous silica, and releases the bactericide, which targets the pathogens at the site of wound infection. Furthermore, the amount of drug released is positively correlated with the severity of infection, allowing for precise control of drug release—more severe infections result in higher drug levels. This avoids drug resistance and side effects caused by indiscriminate drug release, forming a closed-loop "detection-triggered treatment" mechanism.

[0010] (3) Integrated diagnostic and therapeutic system: The detection module (NAFP) and the treatment module (D@MSNs) are uniformly dispersed in a biocompatible dressing matrix to prepare an integrated diagnostic and therapeutic dressing. The dressing matrix has good moisture retention and breathability, can closely adhere to the wound surface, physically block the invasion of external bacteria, and at the same time provide a stable microenvironment for the detection module and the treatment module.

[0011] Preferably, in S11, the concentration of the chloroauric acid aqueous solution is 8-12 mmol / L, and the concentration of the trisodium citrate solution is 35-40 mmol / L; the volume ratio of the chloroauric acid aqueous solution to the trisodium citrate solution is 1:1.

[0012] Preferably, in step S11, heating and stirring continue for 15-50 minutes.

[0013] Preferably, in S12, the concentrations of ribozyme Tz and blocking strand Sc are both 8-12 µM, and the concentration of silver nitrate solution is 1-3 mM; the molar ratio of ribozyme Tz, blocking strand Sc and silver nitrate is 1:2-4:9-11.

[0014] Preferably, in S12, the concentration of signal probe Fp is 8-12 µM; the volume ratio of signal probe Fp, Tz-Ag-Sc and AuNPs solution is 4-6:1-3:90-130.

[0015] Preferably, in S12, the incubation is carried out at 37°C with shaking at 200-500 rpm for 2-5 hours.

[0016] Preferably, in S12, the oscillation is performed at 37°C and a rotation speed of 300-500 rpm for 13-18 hours.

[0017] Preferably, in S21, the concentration of the NaOH aqueous solution is 2.00 mol / L; the ratio of CTAB, NaOH aqueous solution and tetraethyl orthosilicate (TEOS) is 0.3-0.8 g: 3-5 mL: 4-6 mL.

[0018] Preferably, in S22, the ratio of MSNs to 3-chloropropyltrimethoxysilane (ClTMS) is 1-2 g: 1-2 mL.

[0019] Preferably, in S22, the reaction time is 15-25 h.

[0020] Preferably, in S22, the reflux time is 4-8 hours.

[0021] Preferably, in S24, the antibacterial agent includes levofloxacin, and the final concentration of the antibacterial agent is 8-12 mM; the concentration of MSN-N3 in Tris-HNO3 buffer is 1-3 mg / 1-3 mL; the concentration of alkyne-functionalized double-stranded DNA is 18-25 µM; the concentration of CuBr solution is 0.1-0.3 M; and the volume ratio of MSN-N3, alkyne-functionalized double-stranded DNA to CuBr solution is 1-3 mg: 150-250 µL: 1-2 µL.

[0022] Preferably, in S24, the incubation time is 20-30 h.

[0023] Preferably, in step S24, the reaction is carried out at room temperature for 8-20 hours.

[0024] Preferably, in S3, after the NAFP probe and D@MSNs are mixed at a volume ratio of 1:1, the resulting mixture is added to the precursor solution at 8-12% of the mass of the dressing precursor solution, and a calcium chloride solution at a mass fraction of 1-2% is added as a crosslinking agent.

[0025] Preferably, in S3, the mass fraction of the dressing precursor solution is 4-10%, the mass ratio of sodium alginate to sodium carboxymethyl cellulose is 2-4:1-3, and the amount of glycerol added accounts for 1-3% of the mass of the dressing precursor solution.

[0026] A second aspect of the present invention also provides a nucleic acid nanoprobe composite hydrogel prepared by the preparation method described in the first aspect.

[0027] The third aspect of this invention also provides the application of the nucleic acid nanoprobe composite hydrogel described in the second aspect in the preparation of an integrated dressing for the diagnosis and treatment of wound infections.

[0028] Compared with the prior art, the beneficial effects of the present invention are: (1) An innovative three-level tandem controlled-release system of infection biomarker recognition, nucleic acid regulation, and anti-inflammatory drug release was constructed to achieve seamless linkage between detection signal, activation command, and drug release response. Compared with existing independent drug release systems, the tandem mechanism of this invention greatly improves the matching degree between drug release and infection status, fundamentally solving the core defects of disconnect between detection and treatment and imbalance in drug release.

[0029] (2) Precision Treatment: Utilizing the combined mechanism of "nucleic acid molecular lock + diagnostic signal unlock," double-stranded nucleic acid is used as a "smart switch" for drug release. This switch will only open to release anti-inflammatory drugs when a microenvironmental signal of wound infection is detected, and the amount of drug released can be adjusted according to the severity of the infection. This avoids blind drug release from the root cause, improving drug utilization and reducing the side effects of drug-resistant bacteria growth and local skin irritation, fully demonstrating the innovative advantages and application value of this nucleic acid regulation method.

[0030] (3) Strong synergy in integrated diagnosis and treatment: For the first time, a closed-loop system of "nucleic acid sensing detection + signal-triggered drug release" has been established. The detection module and the treatment module can work together seamlessly by relying on the complementary effect of nucleic acid sequences, without the need for additional stimulants or complicated assembly steps. Compared with existing integrated diagnosis and treatment technologies, the working mechanism of this invention is simpler, the response speed is faster (no more than 30 minutes from detection to the start of drug release), and it can also be integrated into dressings, which can provide both physical protection and keep the wound moist. This allows it to be used directly on various wounds such as chronic wounds and acute traumas. It is simple to operate, does not require professional equipment assistance, and has low clinical conversion costs. It is suitable for both hospital clinical use and home daily care, solving the problems of complex processes and difficulty in promotion of existing integrated technologies. Attached Figure Description

[0031] Figure 1 In the image, (A) is a schematic diagram of the structure and response of NAFP; and (B) is a schematic diagram of the preparation of D@MSNs and the tandem controlled-release drug.

[0032] Figure 2 In the image, (A) TEM images of AuNPs and (B) NAFP; (C) Zeta potentials of AuNPs and NAFP; (D) UV absorption spectra of AuNPs and NAFP.

[0033] Figure 3 Fluorescence spectra (A) and real-time fluorescence curves (B) of NAFP under different conditions: red indicates the presence of S. 2- and Ca 2+ When green indicates there is no S 2- Blue indicates that it does not contain Ca. 2+ Purple indicates Ca 2+ and S 2- None of them exist.

[0034] Figure 4 (A) contains different concentrations of S 2- (A) NAFP fluorescence spectrum; (B) Fluorescence intensity as a function of S 2- Standard curve of concentration change.

[0035] Figure 5 This demonstrates the specificity of NAFP's response to different substances in the wound infection environment.

[0036] Figure 6 In the image, (A) is a transmission electron microscope image of mesoporous silica; (B) is a nitrogen adsorption-desorption curve of mesoporous silica; (C) is a pore size distribution diagram of mesoporous silica; and (D) is an X-ray diffraction pattern of mesoporous silica.

[0037] Figure 7 In (A), different concentrations of S were added. 2- (B) The intensity of the LVFX UV characteristic peak in the subsequent solution; 2- The standard curve of the intensity of the UV characteristic peak of the antibacterial drug in the solution.

[0038] Figure 8 The fluorescence signal of the dressing was compared between that applied to a blank culture medium and that inoculated with ordinary Proteus media.

[0039] Figure 9 To count the number of colonies under three different treatment conditions within a circular dressing area with a diameter of 30 mm (three circular areas with a diameter of 30 mm were randomly selected from the culture medium in the undressed group). Detailed Implementation

[0040] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0041] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0042] This invention designs an integrated wound infection treatment dressing based on nucleic acid fluorescent nanoprobes (NAFP) and drug-loaded mesoporous silica (D@MSNs), and further constructs a tandem regulatory system for infection biomarker detection, nucleic acid conformational switching, and on-demand release of anti-inflammatory drugs. The structures of NAFP and D@MSNs, and a schematic diagram of the tandem controlled-release drug are shown below. Figure 1 The implementation plan is as follows: 1. Construction of DNA-gold nanoparticle probe (NAFP) and its performance analysis in detecting wound infection-related sulfides. (1) Gold nanoparticles (AuNPs) with a particle size of approximately 15 nm were synthesized using the sodium citrate reduction method. Targeted ribozyme (Tz), nucleic acid blocking strand (Sc), and fluorescently labeled sensing probe (Fp) were then modified onto the surface of AuNPs via Au-S bonds to prepare the NAFP probe. NAFP was characterized by transmission electron microscopy (TEM), UV-Vis absorption spectroscopy, and Zeta potential. The results showed that the nucleic acid sequence was successfully modified onto the surface of AuNPs, and that NAFP exhibited good dispersibility and structural stability in aqueous solution.

[0043] (2) Sodium sulfide solutions of different concentrations were mixed with CaCl2 solution and NAFP probe. After reacting for 5 minutes, the fluorescence signal was measured (excitation wavelength 495 nm, emission wavelength 520 nm) to evaluate the effect of NAFP on S. 2- Its response capability and detection limit.

[0044] (3) The selectivity of NAFP to potential interfering substances in the wound microenvironment was investigated, including lactic acid (C3H6O3), glutathione (GSH), sodium sulfite (Na2SO3), lipopolysaccharide (LPS), hydrogen peroxide (H2O2), etc., to verify that NAFP has high specificity for H2S and is not affected by common interfering substances.

[0045] 2. Construction of mesoporous silica (D@MSNs) loaded with bactericide and verification of its drug release performance. (1) Mesoporous silica (MSNs) were prepared using hexadecyltrimethylammonium bromide (CTAB) as a template agent and tetraethyl orthosilicate (TEOS) as a silicon source. Azide-modified mesoporous silica (MSNs-N3) was obtained by modification with 3-chloropropyltrimethoxysilane (ClTMS) and N,N-dimethylformamide (DMF). TEM, XRD, and nitrogen adsorption-desorption characterization confirmed that MSNs-N3 had a uniform morphology, a particle size of approximately 80 nm, a pore size of 3.05 nm, and a specific surface area of ​​807.7 m² / g, which is suitable for drug loading and controlled release requirements.

[0046] (2) MSN was reacted with ClTMS in anhydrous toluene to obtain a chloropropyl modified product (MSN-Cl). After CTAB was removed by reflux of ethanol-concentrated hydrochloric acid mixture, it was reacted with sodium azide saturated DMF solution. Subsequently, it was treated with PBS buffer and vacuum dried to obtain azide MSN (MSN-N3).

[0047] (3) Azide-modified mesoporous silica (MSN-N3) was dispersed in a buffer solution. Levofloxacin was selected as the bactericidal model. MSN-N3 was incubated with levofloxacin to achieve drug encapsulation. Alkyne-functionalized double-stranded DNA (H1-H2) was introduced. After centrifugation and washing to remove unbound components, it was finally dispersed in PBS solution to form D@MSNs with double-stranded DNA as the "molecular valve".

[0048] (4) Mix NAFP solution and D@MSNs solution at a 1:1 volume ratio. Take 50 µL of this system solution, add 1 µL of CaCl2 (0.1 M), and then add Na2S solution of different concentrations (0-200 µM). After reacting at room temperature for 20 min, detect the intensity of the specific UV peak of the drug (taking levofloxacin as an example, 293 nm) to evaluate the drug release performance. Test drug release and S 2- The relationship between concentration.

[0049] 3. Preparation and performance verification of therapeutic dressing (nucleic acid nanoprobe composite hydrogel) (1) Mix sodium alginate and sodium carboxymethyl cellulose at a mass ratio of 3:2, add ultrapure water and stir until completely dissolved to prepare a dressing precursor solution. Add an appropriate amount of glycerin (2% by mass) to enhance the moisturizing effect, stir evenly and set aside.

[0050] (2) Mix the NAFP probe and D@MSNs at a volume ratio of 1:1, add 10% of the dressing precursor liquid to the mixing system, ultrasonically disperse for 15 minutes to ensure uniform distribution, then pour into a rectangular mold, add 1% calcium chloride solution as a crosslinking agent, place at room temperature for 30 minutes, demold to obtain the dressing, seal and store at 4℃ for later use.

[0051] 4. Bacterial detection and antimicrobial test for wound infection (1) Select Proteus vulgaris (ATCC33420), a common pathogen causing wound infection, and inoculate it into liquid culture medium. Pour sterilized solid culture medium into a petri dish and let it cool and solidify; take the above bacterial solution and spread it evenly on the surface of the bottom culture medium.

[0052] (2) Antibacterial performance test of dressing: The prepared dressing was cut into circular thin sheets, sterilized by ultraviolet light, and then attached to the surface of a plate. A blank control group (without wound dressing) and a simple dressing control group (without NAFP and D@MSNs) were set up. The petri dishes were placed in a constant temperature incubator at 37℃ for 24 hours, and the diameter of the inhibition zone was observed and measured to evaluate the antibacterial effect.

[0053] (3) Infection detection verification: After the bacteria were cultured in the plate for 6 hours, the integrated diagnostic and therapeutic dressing was attached to the surface of the plate and incubated for 30 minutes. The dressing was then irradiated with a 475 nm excitation light source, and the fluorescence signal intensity was observed by a fluorescence imaging device to verify the in situ detection capability of the dressing for infectious bacteria.

[0054] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0055] Example 1: Preparation and Characterization of NAFP (1) Preparation of NAFP First, gold nanoparticles (AuNPs) were synthesized using the citric acid reduction method. 5 mL of a 10 mmol / L chloroauric acid aqueous solution was mixed with 98 mL of ultrapure water in a three-necked flask. The mixture was then heated to 75°C, and 5 mL of a 38.8 mmol / L trisodium citrate solution was rapidly injected while continuously stirring until the solution turned dark red. The mixture was then heated and stirred for another 20 minutes. Afterward, the solution was washed three times by centrifugation to remove any residual chemicals, finally yielding AuNPs, which were then stored at 4°C.

[0056] Next, NAFP was prepared by mixing the ribozyme (Tz, 10 µM; sequence shown in Table 1) and the blocking strand (Sc, 10 µM; sequence shown in Table 1) at a ratio of 1:3, and then adding 1 mM silver nitrate solution (the molar ratio of Tz, Sc and silver nitrate was 1:3:10). The mixture was then incubated at 37°C with shaking at 300 rpm for 3 hours to obtain the Tz-Ag-Sc complex. Subsequently, 20 µL of Tz-Ag-Sc was mixed with 50 µL of the signal probe (Fp, 10 µM; sequence shown in Table 1) and 1000 µL of AuNPs solution, and the mixture was shaken at 37°C with shaking at 350 rpm for 15 hours to link Tz-Ag-Sc and Fp to the AuNPs surface via Au-S bonds, thus completing the preparation of NAFP.

[0057] Table 1 Nucleic acid sequences used in the experiment (2) TEM, UV and Zeta potential characterization of NAFP Observation using transmission electron microscopy (TEM) revealed ( Figure 2 (A, B) AuNPs are spherical with a diameter of approximately 15 nm. NAFPs maintain a similar morphology and show no significant change in particle size, exhibiting good dispersibility, confirming that DNA probe modification did not disrupt the AuNPs structure. UV-Vis absorption spectroscopy shows ( Figure 2 D), NAFP exhibits a characteristic absorption peak for AuNPs at 522 nm and a characteristic absorption peak for DNA at 260 nm, indicating that the DNA probe has been modified onto the AuNP surface. Zeta potential detection results show ( Figure 2 (C) The Zeta potential of AuNPs was -24.1 mV, while that of NAFP decreased to -28.4 mV, which is due to the modification of the negatively charged DNA strand. This further verifies the successful binding of the DNA probe.

[0058] (3) Feasibility and performance analysis of NAFP for detecting sulfides 1) Feasibility analysis of NAFP for detecting sulfides produced by wound infection Pure water, CaCl2 (0.1 M), Na2S (400 μM), and a mixed solution of CaCl2 and Na2S were added to the NAFP solution, respectively. Figure 3 As shown in (A) and (B), NAFP alone shows almost no fluorescence signal (purple line), but when the cofactor Ca is added... 2+ No fluorescence signal (green line) was generated, indicating that the background signal of NAFP was very low, and Tz activity was completely blocked. However, adding S to NAFP alone... 2- This can cause a slight increase in fluorescence (blue line), while when the target S... 2-and cofactor Ca 2+ When both are present in the system, a fluorescence signal approximately 250 times that of the NAFP background value is immediately generated, reaching 95% fluorescence intensity (red line) after 30 seconds. These results strongly demonstrate that the NAFP response to H₂S depends on S. 2 and Ca 2+ The combined effect of these factors means that Tz activity is effectively activated in the presence of sulfides, and the reaction is rapid and sensitive.

[0059] 2) NAFP Response Performance Evaluation Add 1 μL of 0.1 M CaCl2 and 1 μL of Na2S solution of different concentrations (0~400 μM) to 50 μL of NAFP solution, and measure the fluorescence signal after 5 minutes. The results show ( Figure 4 ), fluorescence intensity and S 2- The concentration showed a good linear relationship: in the concentration range of 0–15 μM, the regression equation was y = 39.4x + 16.9 (R² = 0.998); in the range of 15–100 μM, the regression equation was y = 50.4x - 362.1 (R² = 0.9986), and the limit of detection (LOD) was calculated to be 0.214 μM according to the 3δ / κ rule; when S 2- Concentrations greater than 100 μM, S in NAFP 2- Gradually saturating, fluorescence growth is slow, and the regression equation is y = 2.0x + 4561.7 (R² = 0.982).

[0060] 3) Selective verification of NAFP Take 50 μL of NAFP solution and place it in a centrifuge tube. Add 1 µL of 10 mM lactic acid (C3H6O3), glutathione (GSH), sodium sulfite (Na2SO3), lipopolysaccharide (LPS), hydrogen peroxide (H2O2), and other common substances found in wound infection sites to the NAFP solution, and then detect the fluorescence. Figure 5 The results showed that none of these interfering substances caused obvious fluorescence signals. However, when the interfering substances coexisted with H2S, the fluorescence intensity was 230 times stronger than that of the control group, confirming that NAFP has a specific response to H2S and is not affected by common substances in wounds.

[0061] Example 2: Construction and Performance Verification of D@MSNs 1. Preparation of mesoporous silica (MSNs) Weigh 0.50 g CTAB and dissolve it in 240 mL of ultrapure water. Add 3.5 mL of 2.00 mol / L NaOH aqueous solution and stir thoroughly. Adjust the temperature of the mixture to 80℃, then add 5.00 mL TEOS dropwise and stir vigorously (magnetic stirrer) for 2 h until a white precipitate is formed. Then separate the solid crude product and wash it three times each with ultrapure water and methanol. After vacuum drying, MSNs are obtained.

[0062] 2. Preparation of MSNs-N3 0.5 g MSN was dispersed in 40 mL of anhydrous toluene, and 0.5 mL of ClTMS was added. After reacting for 20 h, the mixture was centrifuged, washed three times with water, and dried under vacuum to obtain chloropropyl-modified MSN (MSN-Cl). To remove the surfactant CTAB, 0.4 g of MSN-Cl particles were weighed and added to a mixed solution of 70 mL of anhydrous ethanol and 0.7 mL of concentrated hydrochloric acid (12 mol / L). After reflux for 6 h, the mixture was centrifuged, washed three times with water, and dried under vacuum at 60 °C overnight.

[0063] Take 0.2 g of purified MSN-Cl and disperse it in 20 mL of DMF solution saturated with sodium azide. Stir at 90 °C for 12 h, centrifuge to separate the obtained particles, then disperse the particles in PBS buffer and stir for 6 h to remove residual N,N-dimethylformamide (DMF) solution in the mesopores. After vacuum drying, obtain azide-treated MSN (MSN-N3).

[0064] 3. Characterization of MSN-N3 Systematic characterization analysis of the prepared MSN-N3 revealed excellent structural properties for drug loading and controlled release. Transmission electron microscopy (TEM) characterization was performed. Figure 6 A) It was confirmed that the mesoporous silica particles had a uniform morphology and a particle size of approximately 80 nm; X-ray diffraction (XRD) tests showed characteristic diffraction peaks of silica, confirming that the prepared material was a silica phase. Figure 6 D); Nitrogen adsorption-desorption experiments showed a specific surface area as high as 807.7 m² / g, with a pore size of 3.05 nm ( Figure 6 B, 6C), the ultra-large specific surface area can significantly improve the encapsulation capacity of drugs, and the appropriate pore size can not only achieve efficient loading of drug molecules, but also provide a smooth channel for subsequent rapid drug release.

[0065] 4. Preparation and Drug Release Performance Evaluation of D@MSNs Weigh 2 mg of MSN-N3 and disperse it in 2 mL of Tris–HNO3 buffer (10 mM, pH 7.2). Add an antibiotic (levofloxacin as an example) to a final concentration of 10 mM. After incubation for 24 h, add 200 µL of 20 µM alkyne-functionalized double-stranded DNA (H1-H2; molar ratio 1:1, sequence shown in Table 2). Then add 1 µL of 0.1 M CuBr solution (solvent: dimethyl sulfoxide: n-butanol = 3:1, v, v). React overnight at room temperature. Centrifuge and wash to remove residual DNA and physically adsorbed antibiotic. Finally, disperse in 2 mL of PBS solution to form D@MSNs with double-stranded DNA as the "molecular valve".

[0066] Table 2 Nucleic acid sequences used in the experiment NAFP solution and D@MSNs solution were mixed at a volume ratio of 1:1. 50 µL of this solution was taken, and 1 µL of 0.1 M CaCl2 was added. Additionally, 1 µL of Na2S solution of different concentrations (0, 5, 10, 15, 20, 40, 60, 80, 100, 150, 200 µM) was added to each tube. The reaction was allowed to proceed at room temperature for 20 min (i.e., the release time), and then the UV spectrum was measured. The results showed (…). Figure 7 The cumulative release of antibacterial drugs showed a good piecewise linear relationship with Na2S concentration: in the concentration range of 0-20µM, the regression equation was y=0.007x+0.097 (R²=0.999), and the release amount in this stage increased with S. 2— The concentration increases slowly, corresponding to a small amount of drug release at low infection levels; within the range of 20–100 µM, the regression equation is y = 0.011x – 0.022 (R² = 0.9962), and the release amount varies with S. 2— The rate of increase in concentration accelerated significantly; when the concentration further increased... 2— When the concentration reaches 100-200 µM, the release of mesoporous silica pores approaches saturation, and the rate of increase in release slows down. The regression equation is y=0.004x+0.715 (R²=0.993).

[0067] Example 3: Construction and Performance Verification of Integrated Therapeutic Dressing 1. Dressing preparation (1) Weigh 3.0 g of sodium alginate and 2.0 g of sodium carboxymethyl cellulose, mix them evenly at a mass ratio of 3:2, add 95 g of ultrapure water, and stir on a magnetic stirrer at room temperature for 6 hours until the solid is completely dissolved to prepare a 5% (w / w) dressing precursor solution. Then add 2.0 g of glycerin (accounting for 2% of the mass of the dressing precursor solution) and continue stirring for 30 minutes until the system is homogeneous and transparent.

[0068] (2) The prepared NAFP probe and D@MSNs were mixed at a volume ratio of 1:1 to obtain a functional nanoparticle hybrid system; (3) Measure 10% of the mass of the dressing precursor solution (before adding glycerin) and add the above mixture to the precursor solution. Place the mixture in an ultrasonic cleaner and ultrasonically disperse for 15 minutes. Slowly pour the evenly dispersed mixture into a rectangular custom mold (5 cm × 3 cm, 2 mm thick). Slowly add 1% calcium chloride solution as a crosslinking agent along the edge of the mold. Let it stand at room temperature for 30 minutes. After the system has completely gelled, gently demold to obtain the integrated diagnostic and therapeutic dressing. Seal it in a sterile sealed bag and store it in a refrigerator at 4°C for later use.

[0069] 2. Preparation of experimental strains Common pathogens causing wound infections, such as Proteus vulgaris (ATCC33420), were selected, inoculated into LB liquid medium, and cultured in a constant temperature shaking incubator at 37°C and 180 rpm for 12 hours for later use.

[0070] 3. Infection detection verification Take a sterile Columbia blood agar plate with a diameter of 90 mm; use a sterile pipette to draw 100 µL of a 1×10⁻⁶ m³ / h blood agar solution. 10 CFU / mL bacterial suspension was evenly spread on the surface of the culture medium. The constructed plate infection model was pre-cultured in a 37℃ incubator for 6 hours (to allow bacteria to produce H2S). Therapeutic dressings were cut into 30 mm diameter circular pieces, sterilized with UV light, and then attached to the plate surface. Incubation continued for 30 minutes. The culture dishes were removed, and the plates were irradiated with a 475 nm excitation light source. The fluorescence signal intensity of the therapeutic dressing was observed using a Gel Smart gel imaging system. A blank plate (with the same therapeutic dressing attached) without bacterial inoculation was set up as a negative control. By comparing the differences in fluorescence signals between the two groups, the in-situ detection capability of the wound dressing for infectious bacteria was verified. Figure 8 The results showed that when the circular dressing was attached to the sterile culture medium, no fluorescence signal was generated under excitation light. After reacting with common Proteus for a period of time, a fluorescence signal was generated within the area of ​​the circular dressing, and the fluorescence signal was stronger in areas where more bacteria were stacked. This indicates that the method can detect sulfides produced by bacteria and thus detect bacteria in situ.

[0071] 4. Antibacterial performance test The concentration after dilution was selected as 1×10. 6CFU / mL bacterial suspension was used, and 30 µL was spread onto Columbia blood agar plates. Prepared circular therapeutic dressings (30 mm in diameter) were sterilized with UV light for 30 minutes (15 minutes on each side) and then affixed to the plates. Three replicates were prepared for each group. Two control groups were also set up: a blank control group (plate infection model only, no dressing) and a blank dressing control group (blank dressing sheets without NAFP and D@MSNs, of the same size and sterilization conditions). All petri dishes were incubated at 37°C for 24 hours. After incubation, the antibacterial rate was calculated using colony counting to evaluate the antibacterial effect of the therapeutic dressing. Figure 9 The results showed that the number of colonies in the dressing group without dressing or without D@MSNs was more than 10. The number of colonies in the blank dressing group was 1-2 fewer than that in the dressing group, which may be because the dressing restricted the growth space of the colonies and inhibited the growth rate. In contrast, the number of colonies in the dressing group containing NAFP and D@MSNs was greatly reduced, with only 0-2 colonies in a circular area with a diameter of 30 mm. This indicates that the dressing has a bactericidal effect on common Proteus bacteria that are commonly found in wound infections.

[0072] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for preparing a tandem controlled-release nucleic acid nanoprobe composite hydrogel, characterized in that, Includes the following steps: S1. Construction of DNA-gold nanoparticle probe NAFP: S11. Synthesis of gold nanoparticles (AuNPs) by citric acid reduction method: Mix chloroauric acid aqueous solution with water, heat to 70-80℃, add trisodium citrate solution until the solution color turns dark red, continue heating and stirring to remove residual chemical substances in the solution, and finally obtain AuNPs solution. S12. Preparation of DNA-gold nanoparticle probe NAFP: The ribozyme strand Tz shown in SEQ ID No. 2 and the blocking strand Sc shown in SEQ ID No. 3 were mixed, and then silver nitrate solution was added. After incubation, the Tz-Ag-Sc complex was obtained. Then, Tz-Ag-Sc was mixed with the signal probe Fp shown in SEQ ID No. 1 and AuNPs solution. By shaking, Au-S bonds were used to connect Tz-Ag-Sc and Fp to the surface of AuNPs, thus obtaining the final product. S2. Constructing mesoporous silica D@MSNs loaded with bactericides: S21. Preparation of mesoporous silica MSNs: CTAB was dissolved in water, and then NaOH aqueous solution was added. The temperature of the mixture was adjusted to 75-85℃, and tetraethyl orthosilicate was added. The reaction was continued until a white precipitate was formed. The solid crude product was then separated, washed and dried to obtain mesoporous silica MSNs. S22. MSNs are dispersed in anhydrous toluene, and then 3-chloropropyltrimethoxysilane is added. After the reaction, chloropropyl-modified MSN, i.e., MSN-Cl, is obtained. Then, MSN-Cl is added to a mixed solution of ethanol and concentrated hydrochloric acid, and then refluxed, centrifuged, washed and dried to remove the surfactant CTAB. S23. The purified MSN-Cl was dispersed in a sodium azide-saturated N,N-dimethylformamide solution and stirred at 85-95 °C for 10-15 h. The resulting particles were then separated by centrifugation. The particles were then dispersed in PBS buffer, stirred to remove residual N,N-dimethylformamide solution from the mesopores, and dried to obtain azide-treated MSN, i.e., MSN-N3. S24. MSN-N3 is dispersed in Tris-HNO3 buffer, antibacterial agent is added, and after incubation, alkyne-functionalized double-stranded DNA as shown in SEQ ID No. 4 and 5 is added. Then CuBr solution is added, and after reaction, residual DNA and physically adsorbed antibacterial agent are removed. Finally, it is dispersed in PBS solution to form D@MSNs with double-stranded DNA as "molecular valve". S3. Preparation of nucleic acid nanoprobe composite hydrogel: Sodium alginate and sodium carboxymethyl cellulose were mixed and dissolved in water to prepare a dressing precursor solution. Glycerin was then added and stirred until the system was homogeneous and transparent. The prepared NAFP probe was mixed with D@MSNs and added to the above precursor solution. The resulting mixture was cross-linked in a mold with calcium chloride solution to obtain the nucleic acid nanoprobe composite hydrogel.

2. The method for preparing a tandem controlled-release nucleic acid nanoprobe composite hydrogel according to claim 1, characterized in that, In S11, the concentration of the aqueous chloroauric acid solution is 8-12 mmol / L, and the concentration of the trisodium citrate solution is 35-40 mmol / L; the volume ratio of the aqueous chloroauric acid solution to the trisodium citrate solution is 1:

1.

3. The method for preparing a tandem controlled-release nucleic acid nanoprobe composite hydrogel according to claim 1, characterized in that, In S12, the concentrations of ribozyme Tz and blocking strand Sc are both 8-12 µM, and the concentration of silver nitrate solution is 1-3 mM; the molar ratio of ribozyme Tz, blocking strand Sc and silver nitrate is 1:2-4:9-11.

4. The method for preparing a tandem controlled-release nucleic acid nanoprobe composite hydrogel according to claim 1, characterized in that, In S12, the concentration of signal probe Fp is 8-12 µM; the volume ratio of signal probe Fp, Tz-Ag-Sc and AuNPs solution is 4-6:1-3:90-130.

5. The method for preparing a tandem controlled-release nucleic acid nanoprobe composite hydrogel according to claim 1, characterized in that, In S21, the concentration of the NaOH aqueous solution is 2.00 mol / L; the ratio of CTAB, NaOH aqueous solution and tetraethyl orthosilicate is 0.3-0.8 g: 3-5 mL: 4-6 mL.

6. The method for preparing a tandem controlled-release nucleic acid nanoprobe composite hydrogel according to claim 1, characterized in that, In S22, the ratio of MSNs to 3-chloropropyltrimethoxysilane is 1-2 g: 1-2 mL.

7. The method for preparing a tandem controlled-release nucleic acid nanoprobe composite hydrogel according to claim 1, characterized in that, In S24, the antibacterial agent includes levofloxacin, and the final concentration of the antibacterial agent is 8-12 mM; the concentration of MSN-N3 in Tris-HNO3 buffer is 1-3 mg / 1-3 mL; the concentration of alkyne-functionalized double-stranded DNA is 18-25 µM, and the concentration of CuBr solution is 0.1-0.3 M; the volume ratio of MSN-N3, alkyne-functionalized double-stranded DNA to CuBr solution is 1-3 mg: 150-250 µL: 1-2 µL.

8. The method for preparing a tandem controlled-release nucleic acid nanoprobe composite hydrogel according to claim 1, characterized in that, In S3, after the NAFP probe and D@MSNs are mixed at a volume ratio of 1:1, the resulting mixture is added to the precursor solution at 8-12% of the mass of the dressing precursor solution, and 1-2% of calcium chloride solution is added as a crosslinking agent.

9. The nucleic acid nanoprobe composite hydrogel prepared by the preparation method according to any one of claims 1-8.

10. The application of the nucleic acid nanoprobe composite hydrogel according to claim 9 in the preparation of an integrated dressing for the diagnosis and treatment of wound infection.

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