A MOF-CQD enzyme-sensitive FRET hydrogel diagnosis and treatment probe and a preparation method and application thereof

Abstract

The application discloses a MOF-CQD enzyme-sensitive FRET hydrogel diagnosis and treatment probe and a preparation method and application thereof, and belongs to the technical field of biomedical detection and treatment. The probe takes carboxyl carbon quantum dots (CQD) as a donor, a metal organic framework (MOF) as an acceptor, and the two are connected through replaceable enzyme response polypeptide bridges. When complete, FRET occurs, and the CQD fluorescence is quenched. After the target enzyme cuts the polypeptide bridge, the FRET is interrupted, the blue / red fluorescence ratio changes, the nanomolar-level quantitative detection is realized, and the self-correction ability is possessed. The probe is packaged in a four-arm PEG transparent hydrogel which does not need a photoinitiator and can be quickly self-crosslinked, and can be made into a patch, a mouthpiece and the like for POCT of oral cavities / skin and the like. After detection, the MOF is activated by 630-660 nm light irradiation to generate active singlet oxygen, local antibacterial or antitumor treatment is realized, and a diagnosis and treatment integrated scheme is constructed.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection and treatment technology, and in particular to a MOF-CQD enzyme-sensitive FRET hydrogel diagnostic probe, its preparation method, and its application. Background Technology

[0002] Fluorescence resonance energy transfer (FRET) is widely used in biomedical detection and imaging due to its high sensitivity and selectivity. However, traditional probes often use small-molecule organic dyes or noble metal nanoparticles as acceptors, which often suffer from poor fluorescence stability, general biocompatibility, easy photobleaching, and high background noise, limiting their continued application in clinical settings.

[0003] Carbon quantum dots (CQDs) are considered ideal FRET donors due to their good water solubility, excellent biocompatibility, high fluorescence quantum yield, and resistance to photobleaching. However, existing CQD probes still face two major bottlenecks: (1) a lack of strong quencher acceptors that efficiently overlap with their emission spectra, resulting in limited signal change amplitude in applications such as enzyme responses; (2) most systems exist in solution form, which is not conducive to portability and standardized operation, especially lacking transparent, soft, and malleable solid (or gel) carriers to achieve stable fixation and easy use.

[0004] Metal-organic frameworks (MOFs) are rapidly developing in the biomedical field due to their designable structure, tunable pores, large specific surface area, and good biocompatibility. Taking Zr-based PCN-224 as an example, it has a broad absorption band around 430 nm and can efficiently generate reactive singlet oxygen under light irradiation, possessing natural potential for photodynamic therapy (PDT). However, many MOF materials are prone to collapse in aqueous solutions, and the structure of some MOF materials may decompose at high temperatures, thus affecting their performance and lifespan. Summary of the Invention

[0005] This invention aims to construct a MOF-CQD enzyme-sensitive FRET hydrogel diagnostic probe, its preparation method and application, and proposes corresponding preparation processes and application schemes to improve enzyme response sensitivity and signal-to-noise ratio, thereby realizing the integration of detection and treatment.

[0006] To achieve the above objectives, the present invention provides the following solution: In a first aspect, the present invention provides a method for preparing a MOF-CQD enzyme-sensitive FRET hydrogel diagnostic probe, the method comprising the following steps: CQDs Synthesis and Surface Carboxylation Modification: CQDs were synthesized by high-temperature thermal method, and the surface of CQDs was modified by carboxyl-containing ligands to obtain modified CQDs; Preparation of Ag-modified MOFs: H2TCPP, ZrOCl2·8H2O and benzoic acid were dissolved in DMF, stirred, centrifuged and washed to obtain MOF nanoparticles; MOFs were dispersed in water, AgNO3 was added, ultrasonically mixed, stirred and NaBH4 solution was added, centrifuged and washed to obtain Ag-modified MOFs; Construction of FRET probe: Synthesize a peptide chain that recognizes and cleaves the enzyme sequence; chemically modify both ends of the peptide chain, introducing an amino group at one end and a thiol group at the other end; connect the modified CQDs, peptide chain and MOF obtained above to form a FRET probe; Preparation of nanohydrogel probes: The obtained FRET probes are added at a concentration of 50-150 μg / mL to a polyethylene glycol solution with maleimide at the end or a polyethylene glycol solution with thiol at the end. The two polyethylene glycol solutions are then mixed and injected into a mold for curing to obtain nanohydrogel probes.

[0007] Preferably, the sequence of the peptide chain is as shown in SEQ ID NO.1.

[0008] Preferably, 50-150 mg of H2TCPP (0.1-0.15 mM), 100-500 mg of ZrOCl2·8H2O (0.93-1.0 mM), and 2.6-3.0 g of benzoic acid (20-25 mM) are dissolved in 50-150 mL of DMF. The solution is stirred at 100-500 r / min for 3-8 h at 80-100 °C, centrifuged at 10000-20000 r / min for 10-50 min, and washed to obtain MOF nanoparticles.

[0009] Preferably, 100 mg of 0.13 mM H2TCPP, 300 mg of 0.93 mM ZrOCl2·8H2O, and 2.8 g of 23 mM benzoic acid are dissolved in 100 mL of DMF, stirred at 300 r / min for 5 h at 90 °C, centrifuged at 15000 r / min for 30 min, and washed to obtain MOF nanoparticles.

[0010] Preferably, 10-90 mg of the prepared MOF is dispersed in 10-90 mL of water for 10-30 min, 100-200 μL of AgNO3 with a concentration of 0.05-0.15 M is added, the mixture is ultrasonically mixed for 10-30 min, stirred at 600-1000 rpm, and 3.5-4.0 mL of NaBH4 solution with a concentration of 0.05-0.15 M is added. The mixture is stirred for 0.5-1.5 h, centrifuged, and washed to obtain the Ag-modified MOF.

[0011] Preferably, 50 mg of the prepared MOF is dispersed in 50 mL of water for 20 min, 150 μL of 0.1 M AgNO3 is added, the mixture is ultrasonically mixed for 20 min, stirred at 800 rpm and 3.75 mL of 0.1 M NaBH4 solution is added, stirred for 1 h, centrifuged and washed to obtain Ag-modified MOF.

[0012] Preferably, the modified QD, MOF and peptide chain are linked in a molar ratio of 10:10:1 to obtain the FRET probe; The concentrations of the polyethylene glycol solutions with maleimide-terminated and those with thiol-terminated are both 7%; the FRET probe is added to either the maleimide-terminated or thiol-terminated polyethylene glycol solutions at a concentration of 100 μg / mL.

[0013] Secondly, the present invention also provides a hydrogel diagnostic probe prepared by the preparation method described above.

[0014] Thirdly, the present invention also provides a product for detecting Porphyromonas gingivalis, the product comprising the aforementioned hydrogel diagnostic probe.

[0015] Fourthly, the present invention also provides a product for inhibiting the growth of Porphyromonas gingivalis, the product comprising the aforementioned hydrogel diagnostic probe.

[0016] Preferably, the detection and treatment of bacterial infections such as periodontitis is a detection and treatment drug.

[0017] The present invention discloses the following technical effects: 1) Construction of the FRET detection unit: Specifically, carbon quantum dots (QDs) are used as the fluorescent donor, and nano-MOFs with fluorescence quenching capability are used as the fluorescent acceptor. By designing peptide chains containing enzyme cleavage sites as linkers, QDs are coupled to nano-MOFs to form a fluorescent probe. In the absence of the analyte enzyme, the fluorescence of QDs is effectively quenched by the nano-MOFs due to the FRET effect. When the analyte enzyme is present in the system, the enzyme can specifically cleave the peptide chain linking QDs and nano-MOFs, causing QDs to separate from the nano-MOFs, relieving the FRET effect, and restoring and enhancing the fluorescence signal of QDs. By detecting the intensity of the restored fluorescence signal, rapid, sensitive, and specific enzyme detection can be achieved.

[0018] 2) Achieving Photodynamic Therapy Function: This invention utilizes the photodynamic ability of the nano-MOF material itself to generate singlet oxygen under specific light irradiation. When the hydrogel system encapsulating this MOF is irradiated at a specific wavelength (660 nm), the MOF can be excited and generate singlet oxygen with strong oxidizing power. Singlet oxygen can efficiently kill bacteria, destroy their cell structure, inhibit biofilm formation, and has anti-inflammatory effects, providing a new approach to solving the problem of traditional antibiotic resistance.

[0019] 3) Optimized hydrogel carrier: The aforementioned FRET detection unit and photodynamic MOF nanoparticles are co-encapsulated in an optimized transparent polyethylene glycol (PEG) hydrogel. The PEG hydrogel possesses excellent biocompatibility, high transparency, and a tunable pore structure, ensuring probe stability, biomolecule diffusion (such as enzyme-peptide chain contact), and light penetration. By controlling the cross-linking structure and mechanical strength of the hydrogel, various insertable, adhesive, or injectable forms can be prepared, suitable for multiple local sites on the body surface or in vivo, achieving highly sensitive detection of target enzymes and localized targeted therapy.

[0020] Ease of use: It is easy to operate, requires no complicated equipment or professional personnel, and can be performed in clinical or home settings.

[0021] High sensitivity: Utilizing the sensitivity of the FRET effect, it is possible to detect low concentrations of target enzymes.

[0022] Specificity: Based on the specific recognition of enzymes, false positive results are reduced.

[0023] Stability: The transparent PEG hydrogel system provides a stable environment for detection, improving the reliability and reproducibility of the detection results.

[0024] In summary, FRET diagnostic and therapeutic probes constructed using the CQDs / MOF composite system can provide comprehensive solutions for improving signal-to-noise ratio, enhancing photostability, and achieving therapeutic functions, thus offering a new pathway for the development of clinically translatable biological probes. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention.

[0026] Figure 1 The images show the absorption and emission spectra of the modified QD; where a is the absorption spectrum and b is the emission spectrum. Figure 2 TEM observation results (a) and EDS energy dispersive spectroscopy analysis results (b); Figure 3SEM image of the nanohydrogel probe; Figure 4 The figure shows the stress-strain curve of the nano-hydrogel probe; where a represents the mechanical strength test of the hydrogel and b represents the optical transparency test of the nano-hydrogel probe. Figure 5 The graph shows the concentration response of the nanohydrogel probe; where a is the fluorescence image obtained under 365nm excitation; the enzyme concentrations from right to left are 0nM, 100nM, 200nM, 500nM, 8000nM, and 1000nM; b is the fluorescence emission spectrum measured by a spectrometer. Figure 6 The standard curve for the concentration of Pg pathogenic enzymes; Figure 7 The specific response curves of the nanohydrogel probes to different bacteria are shown. Figure 8 The graph shows the effect of different concentrations of FRET probe on cell viability; where a is a Live / Dead staining image of cells after treatment with different concentrations of FRET probe (green represents surviving cells, red / black represents dead cells; scale bar 50µm); b is the statistical data of cell viability under the corresponding conditions; Figure 9 These are representative curves generated by ROS under illumination conditions; Figure 10 The image shows the in vitro antibacterial effect; where a represents the colony count of different treatment groups and b represents the biofilm OD value of different treatment groups. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Example 1: Preparation of FRET probe and photodynamic nano-MOF This embodiment aims to prepare FRET probes for use in therapeutic systems and elaborates in detail the synthesis and functionalization of nanometal-organic frameworks (MOFs) with intrinsic photodynamic effects.

[0033] 1) Synthesis and surface carboxylation modification of carbon quantum dots (CQDs) Carbon quantum dots (CQDs) were prepared by a hydrothermal method: 1.0 g of citric acid was dissolved in 10 mL of deionized water, transferred to a polytetrafluoroethylene reactor, and reacted at 180 °C for 4 h. After cooling, the solution was dialyzed through a 10 kDa dialysis bag for 24 h to obtain a CQDs solution. Subsequently, surface carboxylation was performed: 10 mL of CQDs (5 mg / mL) was taken... -1 Add 50 mg of mercaptopropionic acid, adjust the pH to 8-10, and stir at room temperature for 12 h to complete ligand exchange. Dialyze again to remove free ligands, yielding CQDs with carboxyl-rich surfaces. Transmission electron microscopy (TEM) revealed that the modified CQDs had a spherical nanomorphology with an average diameter of 5 nm. Optical properties were characterized using fluorescence spectroscopy; the absorption and emission spectra are as follows. Figure 1 As shown in Figure ab, CQDs exhibit a significant absorption peak at approximately 340-360 nm, with absorption rapidly decaying above 400 nm. Under 365 nm excitation, they show a strong single-peak emission at approximately 410-430 nm, exhibiting a large Stokes shift, which helps reduce self-absorption and improve the signal-to-noise ratio. Carboxylation treatment did not significantly alter their optical properties.

[0034] 2) Preparation of Ag-modified MOFs H₂TCPP (100 mg, 0.13 mM), ZrOCl₂·8H₂O (300 mg, 0.93 mM), and benzoic acid (2.8 g, 23 mM) were dissolved in 100 mL of DMF in a 250 mL round-bottom flask, and the mixture was stirred at 300 r / min for 5 h at 90 °C. After the reaction was complete, MOF nanoparticles were collected by centrifugation (15000 r / min, 30 min) and then washed three times with fresh DMF.

[0035] To load silver nanoparticles onto the surface of MOF, a reduction method was employed in this invention. First, 50 mg of the prepared MOF was uniformly dispersed in 50 mL of water for 20 min by ultrasonic treatment. Next, 150 μL of 0.1 M AgNO3 was added, and the mixture was ultrasonically mixed for 20 min. Then, the mixture was transferred to a magnetic stirrer and stirred at 800 rpm. During stirring, freshly prepared NaBH4 solution (0.1 M, 3.75 mL) was rapidly added, and the stirring speed was maintained at 800 rpm for 1 h. The sample was centrifuged at 6000 rpm and washed three times.

[0036] TEM observation revealed that Ag nanoparticles were deposited on the MOF framework, such as Figure 2 As shown in Figure a; EDS energy dispersive spectroscopy further confirmed the presence of Ag elements in these nanoparticles, indicating that Ag NPs were successfully loaded onto the MOF, as shown in Figure a. Figure 2 As shown in b.

[0037] 3) Synthesis of peptide chains and construction of FRET probes Prepare a polypeptide linker that can be recognized and cleaved by the target enzyme. Synthesize a peptide chain that recognizes and cleaves an arginine-specific cysteine ​​protease (Rgp) sequence: GGFLVRGC (SEQ ID NO.1).

[0038] The peptide chain contains specific enzyme cleavage sites, and both ends are chemically modified: an amino group is introduced at one end to couple with the carboxyl group on the surface of CQDs; and a thiol group is introduced at the other end to couple with the silver nanoparticles on the surface of the nano MOF.

[0039] Modified CQDs, modified peptide chains, and modified MOFs were linked in an optimized molar ratio of 10:10:1 to form a FRET probe with a CQDs-peptide-MOF ternary structure. The linkage method involved first connecting carboxylated quantum dots to the amino terminus of the polypeptide chain via an amino coupling reaction, and then coupling the polypeptide chain's thiol groups to pre-defined binding sites (silver-sulfur bond sites) on the MOF surface.

[0040] Example 2: Preparation and Encapsulation of Nanohydrogel Probes This embodiment aims to elaborate in detail the preparation method of the optimized hydrogel carrier, and the encapsulation of FRET probes and photodynamic nano-MOFs in the hydrogel.

[0041] 1) Preparation of PEG hydrogel solution Weigh appropriate amounts of maleimide-terminated polyethylene glycol (PEG-MAL, 10 kDa) and thiol-terminated polyethylene glycol (PEG-SH, 3 kDa), and dissolve them separately in HEPES buffer (pH 7.2) to prepare solutions of a specific concentration (7%, w / v). Ensure the molar ratio of maleimide groups to thiol groups is 1:1 during solution preparation to facilitate efficient cross-linking.

[0042] 2) Encapsulation and hydrogel molding of FRET probes The FRET probe prepared in Example 1 was added to the above-mentioned PEG-MAL solution or PEG-SH solution at an optimized concentration of 100 μg / mL to ensure uniform dispersion of the nanoparticles in the solution. Subsequently, the two PEG solutions were mixed thoroughly. The mixed solution containing nanoparticles was rapidly injected into a pre-designed mold. At room temperature, PEG-MAL and PEG-SH rapidly crosslinked via a Michael addition reaction, forming and solidifying a hydrogel network, thereby effectively encapsulating the nanotherapeutic particles within it. The hydrogel solidification time was completed within 5 minutes. The solidified hydrogel was removed from the mold and thoroughly washed with HEPES solution to remove unreacted substances, thus obtaining the nanohydrogel probe.

[0043] The performance characteristics of the prepared nanohydrogel probes were characterized by observing the internal pore structure of the hydrogel using scanning electron microscopy (SEM). The results are as follows: Figure 3 As shown, the average pore size of the nanohydrogel probe is 500 nm, which ensures favorable diffusion of enzymes, substrates, and singlet oxygen. The mechanical strength of the hydrogel was tested using a universal testing machine, and the results are as follows... Figure 4 As shown in Figure a, this figure indicates that the stress of the hydrogel increases nearly linearly with strain during stretching, and fractures at approximately 240% strain, with a limiting stress of about 0.9 MPa. This demonstrates its high ductility and good mechanical strength, as well as its uniform network structure and stable cross-linking. Simultaneously, the transparency of the nano-hydrogel probe was observed using a UV-Vis spectrophotometer. Figure 4 (b) The test results showed that the transmittance of the nano-hydrogel probe reached over 97% in the wavelength range of 400-800 nm, which ensured that it did not affect the reading of fluorescence signals during detection and the light transmission during photodynamic therapy.

[0044] Example 3: Detection and Photodynamic Therapy Function Verification of Nanohydrogel Probes This embodiment aims to verify the function and effect of nanohydrogel probes in detecting and performing photodynamic therapy, using the pathogenic enzyme of Porphyromonas gingivalis, gingival protease, as an example.

[0045] 1. Detection performance verification 1) Concentration response and sensitivity Different concentrations (0-1000 nM) of purified *Porphyromonas gingivalis* pathogenic enzyme solutions were used as test samples and added dropwise to the nanoprobe containing the FRET probe prepared in this invention. After incubation for a specific time of 10 min, the fluorescence signal of the nanoprobe was measured using a fluorescence spectrometer. The increase in fluorescence intensity in the QD (blue light region) and the decrease in fluorescence intensity in the MOF (red light region) were recorded to construct a standard curve of fluorescence signal ratio (e.g., blue / red light intensity ratio) versus Pg pathogenic enzyme concentration, in order to determine the detection sensitivity, limit of detection (LOD), and linear range of the nanohydrogel probe.

[0046] The results are as follows Figure 5 As shown in Figure ab, this illustrates the fluorescence signal changes of the nanohydrogel probe described in this invention at different concentrations of pathogenic enzymes from *Porphyromonas gingivalis*. With increasing pathogenic enzyme concentration, the signal in the red region, representing MOF fluorescence emission intensity, decreases, while the signal in the blue region, representing carbon quantum dot fluorescence emission intensity, significantly increases. This result strongly validates the excellent concentration-dependent response and high sensitivity of the nanohydrogel probe described in this invention to the target pathogenic enzyme, and further suggests its potential for ratiometric fluorescence detection, thereby improving detection accuracy and anti-interference capabilities. Based on the constructed standard curve (e.g....), Figure 6 As shown in the figure, the LOD of the nanohydrogel probe is 5 nM, which can meet clinical needs.

[0047] 2) Detection specificity The nanoprobes prepared in this invention were exposed to equal concentrations (10) 2 10 3 10 4 10 5 10 6 10 7 10 8 Porphyromonas gingivalis (per / mL) P. gingivalis ) and other common oral bacteria such as Fusobacterium nucleatum ( F. nucleatum ), oral streptococci ( Streptococcus oralis ) and Focusae ( T. forsythia The culture medium or lysis buffer was incubated for 10 min, and the fluorescence signal was measured. The specific response of this system to the Pg pathogenic enzyme was verified, and its cross-reactivity with non-target bacteria was evaluated.

[0048] The experimental results are shown in Figure 7 , Figure 7 The data shows that Porphyromonas gingivalis (Porphyromonas gingivalis) P. gingivalis Concentration 10 4 Up to 10 5 Within the per mL range, the fluorescence signal at 420 nm increased sharply and reached saturation at higher concentrations. However, for the control *Fusobacterium nucleatum* (…),… F. nucleatum ), oral streptococci ( Streptococcus oralis ) and Focusae ( T. forsythia Even at similar or even higher bacterial concentrations, the detection signal remained consistently low. This fully demonstrates that the nano-hydrogel probe prepared in this invention possesses excellent specific recognition ability for *Porphyromonas gingivalis*, effectively distinguishing the target pathogen from other common bacteria in the oral cavity, thus providing a reliable basis for accurate diagnosis.

[0049] 2. Biosafety testing In in vitro cell experiments, this invention evaluated the effect of FRET probes at different concentration gradients (20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL) on the survival rate of gingival fibroblasts, with PBS solution as the control group.

[0050] Experimental results are as follows Figure 8 As shown in Figures ab, the survival rate of gingival fibroblasts was higher than 98% in the PBS control group and at FRET probe concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. This result clearly indicates that the FRET probe has no significant cytotoxicity to gingival fibroblasts within these tested concentration ranges, demonstrating good biocompatibility. Therefore, this invention ultimately selected 100 μg / mL as the working concentration of the FRET probe for subsequent bioassay and therapeutic experiments. This concentration was chosen to maximize its potential efficacy in detection and treatment while maintaining high biocompatibility.

[0051] 3. Validation of photodynamic therapy function 1) Singlet oxygen production capacity The MOF-encapsulated nanohydrogel probe was exposed to light of a specific wavelength and intensity (660 nm, optical power density 30 mW / cm²) for 20 min. The amount of singlet oxygen generated in the solution was detected by a specific singlet oxygen fluorescent probe to demonstrate its photodynamic effect.

[0052] The experimental results are as follows Figure 9As shown in the figure, this illustrates the typical response of the probe generating reactive oxygen species (ROS) under illumination. A distinct set of positive and negative peaks appears each time the light is turned on, and the signal returns to near the baseline after the light is turned off, indicating that ROS generation is light-triggered, reversible, and repeatable.

[0053] 2) In vitro antibacterial effect A biofilm model or suspension of *Porphyromonas gingivalis* was constructed in vitro. A nanohydrogel probe containing MOF was placed within it and subjected to 20 min of light irradiation (light parameters: wavelength 660 nm, light power density 10-50 mW / cm²). 2 Simultaneously, PBS buffer and dark treatment with nanohydrogel probes served as two controls. Bacterial survival or biofilm inhibition / clearance rate was assessed by colony counting and biofilm amount (crystal violet staining).

[0054] Experimental results are as follows Figure 10 As shown in Figure ab, the experimental results in this figure confirm the photodynamic antibacterial effect of MOF encapsulated in the nanohydrogel probe. The nanohydrogel probe prepared using this invention can effectively reduce the number of Porphyromonas gingivalis colonies. Figure 10 (a), while also exhibiting high inhibition / scavenging rates on biofilms ( Figure 10 (b) This means that the nanohydrogel probe has excellent in vitro antibacterial effect.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a MOF-CQD enzyme-sensitive FRET hydrogel diagnostic probe, characterized in that, The preparation method includes the following steps: CQDs Synthesis and Surface Carboxylation Modification: CQDs were synthesized by high-temperature thermal method, and the surface of CQDs was modified by carboxyl-containing ligands to obtain modified CQDs; Preparation of Ag-modified MOFs: H2TCPP, ZrOCl2·8H2O and benzoic acid were dissolved in DMF, stirred, centrifuged and washed to obtain MOF nanoparticles; MOFs were dispersed in water, AgNO3 was added, ultrasonically mixed, stirred and NaBH4 solution was added, centrifuged and washed to obtain Ag-modified MOFs; Construction of FRET probe: Synthesize a peptide chain that recognizes and cleaves the enzyme sequence; chemically modify both ends of the peptide chain, introducing an amino group at one end and a thiol group at the other end; connect the modified CQDs, peptide chain and MOF obtained above to form a FRET probe; Preparation of nanohydrogel probes: The obtained FRET probes are added at a concentration of 50-150 μg / mL to a polyethylene glycol solution with maleimide at the end or a polyethylene glycol solution with thiol at the end. The two polyethylene glycol solutions are then mixed and injected into a mold for curing to obtain nanohydrogel probes.

2. The preparation method according to claim 1, characterized in that, The sequence of the peptide chain is shown in SEQ ID NO.

1.

3. The preparation method according to claim 1, characterized in that, The following steps were performed: 50-150 mg of 0.1-0.15 mM H2TCPP, 100-500 mg of 0.93-1.0 mM ZrOCl2·8H2O, and 2.6-3.0 g of 20-25 mM benzoic acid were dissolved in 50-150 mL of DMF. The solution was stirred at 100-500 r / min for 3-8 h at 80-100 °C, centrifuged at 10000-20000 r / min for 10-50 min, and washed to obtain MOF nanoparticles.

4. The preparation method according to claim 3, characterized in that, 100 mg of 0.13 mM H2TCPP, 300 mg of 0.93 mM ZrOCl2·8H2O, and 2.8 g of 23 mM benzoic acid were dissolved in 100 mL of DMF. The solution was stirred at 300 r / min for 5 h at 90 °C, centrifuged at 15000 r / min for 30 min, and washed to obtain MOF nanoparticles.

5. The preparation method according to claim 3, characterized in that, The prepared MOF was dispersed in 10-90 mg of water for 10-30 min, 100-200 μL of 0.05-0.15 M AgNO3 was added, and the mixture was ultrasonically mixed for 10-30 min. The mixture was stirred at 600-1000 rpm and 3.5-4.0 mL of 0.05-0.15 M NaBH4 solution was added. The mixture was stirred for 0.5-1.5 h, centrifuged, and washed to obtain the Ag-modified MOF.

6. The preparation method according to claim 5, characterized in that, 50 mg of the prepared MOF was dispersed in 50 mL of water for 20 min, 150 μL of 0.1 M AgNO3 was added, and the mixture was ultrasonically mixed for 20 min. The mixture was stirred at 800 rpm and 3.75 mL of 0.1 M NaBH4 solution was added. The mixture was stirred for 1 h, centrifuged, and washed to obtain the Ag-modified MOF.

7. The preparation method according to claim 1, characterized in that, The modified CQDs, peptide chains and MOFs are linked together in a molar ratio of 10:10:1 to obtain the FRET probe. The concentrations of the polyethylene glycol solutions with maleimide-terminated and those with thiol-terminated are both 7%; the FRET probe is added to either the maleimide-terminated or thiol-terminated polyethylene glycol solutions at a concentration of 100 μg / mL.

8. A hydrogel diagnostic probe prepared by the preparation method according to any one of claims 1-7.

9. A product for detecting Porphyromonas gingivalis, characterized in that, The product comprises the hydrogel diagnostic probe as described in claim 8.

10. A product that inhibits the growth of *Porphyromonas gingivalis*, characterized in that, The product comprises the hydrogel diagnostic probe as described in claim 8.

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