MOF-CQD enzyme-sensitive FRET hydrogel diagnosis and treatment probe as well as preparation method and application thereof

Abstract

The invention discloses an MOF-CQD enzyme-sensitive FRET hydrogel diagnosis and treatment probe as well as a preparation method and application thereof, and belongs to the technical field of biomedical detection and treatment. The probe takes a carboxyl carbon quantum dot (CQD) as a donor and a metal organic framework (MOF) as an acceptor, and the donor and the acceptor are connected through a replaceable enzyme response polypeptide bridge; when the fluorescence is complete, FRET is generated to quench the CQD fluorescence, the FRET is interrupted after the target enzyme cuts off the peptide bridge, and the blue / red fluorescence ratio is changed to realize nanomole-level quantitative detection and has self-correction capability. 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 prepared into a film, a tooth socket and the like for POCT (point-of-care testing) of parts such as an oral cavity / skin and the like. After detection, the MOF is activated by illumination of 630-660 nm to generate active singlet oxygen, so that local antibacterial or anti-tumor treatment is realized, and a diagnosis and treatment integrated scheme is constructed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical detection and treatment, in particular to a MOF-CQD enzyme-sensitive FRET hydrogel diagnosis and treatment probe and a preparation method and application thereof. BACKGROUND

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

[0003] Carbon quantum dots (CQDs) are considered as 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) lack of strong quenching acceptors with efficient spectral overlap, resulting in limited signal change range in enzyme response applications; (2) most systems exist in solution form, which is not conducive to portable and standardized operation, especially lacking transparent, soft, and shapeable solid (or gel) carriers to achieve stable fixation and easy use.

[0004] Metal-organic frameworks (MOFs) have rapidly developed in the field of biomedicine due to their designable structure, adjustable pore size, large specific surface area, and good biological safety. For example, Zr-based PCN-224 has a wide absorption band near 430 nm and can efficiently generate reactive singlet oxygen under light, which has natural photodynamic therapy (PDT) potential. However, many MOF materials can collapse in aqueous solution, and the structure of some MOF materials may decompose at high temperatures, affecting their performance and service life. SUMMARY

[0005] The present application aims to construct a MOF-CQD enzyme-sensitive FRET hydrogel diagnosis and treatment probe and its preparation method and application, and proposes corresponding preparation process and application scheme, so as to improve the enzyme response sensitivity and signal-to-noise ratio, and realize the integration of detection and treatment.

[0006] To achieve the above-mentioned purpose, the present application provides the following scheme:

[0007] In a first aspect, the present application provides a preparation method of a MOF-CQD enzyme-sensitive FRET hydrogel diagnosis and treatment probe, which comprises the following steps:

[0008] CQDs synthesis and surface carboxylation modification: high-temperature thermal synthesis of CQDs, and modification of the surface of CQDs with carboxyl-containing ligands to obtain modified CQDs;

[0009] Preparation of MOF modified by Ag: H2TCPP, ZrOCl2.8H2O and benzoic acid were dissolved in DMF, MOF nanoparticles were obtained by stirring, centrifugation and washing; the MOF was dispersed in water, AgNO3 was added, ultrasonic mixing, stirring and adding NaBH4 solution, centrifugation and washing to obtain MOF modified by Ag;

[0010] Construction of FRET probe: the peptide chain recognizing and cutting enzyme sequence was synthesized; the peptide chain was chemically modified at both ends, one end introduced amino group and the other end introduced thiol group; the modified CQDs, the peptide chain and the MOF were connected to form the FRET probe;

[0011] Preparation of nanohydrogel probe: the obtained FRET probe was added to the polyethylene glycol solution with maleimide at the end or the polyethylene glycol solution with thiol at the end at a concentration of 50-150 μg / mL, then the two polyethylene glycol solutions were mixed, injected into the mold and solidified to obtain the nanohydrogel probe.

[0012] Preferably, the sequence of the peptide chain is shown in SEQ ID NO. 1.

[0013] Preferably, 50-150 mg of H2TCPP with a concentration of 0.1-0.15 mM, 100-500 mg of ZrOCl2.8H2O with a concentration of 0.93-1.0 mM and 2.6-3.0 g of benzoic acid with a concentration of 20-25 mM were dissolved in 50-150 mL of DMF, stirred at a speed of 100-500 r / min at 80-100 ℃ for 3-8 h, centrifuged at 10,000-20,000 r / min for 10-50 min, and washed to obtain MOF nanoparticles.

[0014] Preferably, 100 mg of H2TCPP with a concentration of 0.13 mM, 300 mg of ZrOCl2.8H2O with a concentration of 0.93 mM and 2.8 g of benzoic acid with a concentration of 23 mM were dissolved in 100 mL of DMF, stirred at a speed of 300 r / min at 90 ℃ for 5 h, centrifuged at 15,000 r / min for 30 min, and washed to obtain MOF nanoparticles.

[0015] Preferably, 10-90 mg of the prepared MOF was 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 was added, ultrasonic mixing was performed for 10-30 min, stirring was performed at 600-1,000 rpm and 3.5-4.0 mL of NaBH4 solution with a concentration of 0.05-0.15 M was added, stirring was performed for 0.5-1.5 h, centrifugation and washing were performed to obtain MOF modified by Ag.

[0016] Preferably, the MOF prepared by taking 50 mg is dispersed in 50 mL of water for 20 min, 150 μL of 0.1 M AgNO3 is added, ultrasonic mixing is performed for 20 min, stirring is performed at 800 rpm and 3.75 mL of 0.1 M NaBH4 solution is added, stirring is performed for 1 h, centrifugation and washing are performed to obtain the Ag-modified MOF.

[0017] Preferably, the modified QD, MOF and peptide chain are connected in a molar ratio of 10:10:1 to obtain the FRET probe.

[0018] The concentration of the maleimide-terminated polyethylene glycol solution and the thiol-terminated polyethylene glycol solution is 7%; the FRET probe is added to the maleimide-terminated polyethylene glycol solution or the thiol-terminated polyethylene glycol solution at a concentration of 100 μg / mL.

[0019] In a second aspect, the present application further provides a hydrogel diagnosis and treatment probe prepared by the preparation method.

[0020] In a third aspect, the present application further provides a product for detecting Porphyromonas gingivalis, wherein the product comprises the hydrogel diagnosis and treatment probe.

[0021] In a fourth aspect, the present application further provides a product for inhibiting the growth of Porphyromonas gingivalis, wherein the product comprises the hydrogel diagnosis and treatment probe.

[0022] Preferably, the detection and treatment of bacterial infections such as periodontitis are for detection and treatment drugs.

[0023] The present application discloses the following technical effects:

[0024] 1) Constructing a FRET detection unit: Specifically, carbon quantum dots (QD) are used as a fluorescence donor, and a nano MOF with fluorescence quenching ability is used as a fluorescence acceptor. A peptide chain containing a restriction enzyme site is designed as a connecting arm to couple QD and nano MOF, forming a fluorescence probe. In the absence of the enzyme to be detected, the fluorescence of QD is effectively quenched by nano MOF due to the FRET effect. When the enzyme to be detected exists in the system, the enzyme can specifically cut the peptide chain connecting QD and nano MOF, causing QD and nano MOF to separate, the FRET effect is removed, and the fluorescence signal of QD is restored and enhanced. By detecting the recovery intensity of the fluorescence signal, rapid, sensitive and specific detection of the enzyme can be achieved.

[0025] 2) Realize the function of photodynamic therapy: The application utilizes the photodynamic ability of nano MOF material itself to generate singlet oxygen under specific light. When the hydrogel system encapsulating the MOF is irradiated under specific wavelength (660 nm), the MOF can be excited and generate singlet oxygen with strong oxidizing ability. Singlet oxygen can efficiently kill bacteria, destroy their cell structure, inhibit biofilm formation, and has anti-inflammatory effect, providing a new way for traditional antibiotic resistance problem.

[0026] 3) Optimize the hydrogel carrier: The above-mentioned FRET detection unit and nano MOF with photodynamic effect are jointly encapsulated in the optimized transparent polyethylene glycol (PEG) hydrogel. PEG hydrogel has good biocompatibility, high transparency and adjustable pore size structure, ensuring the stability of the probe, the diffusion of biological molecules (such as the contact of enzymes and peptide chains) and the penetration of light. With the controllable adjustment of the cross-linking structure and mechanical strength of the hydrogel, various forms of insertion, application or injection can be prepared, which is suitable for various local sites on the body surface or in the body, realizing high sensitivity detection and local site-specific treatment of target enzymes.

[0027] Convenience: simple operation, no need for complex equipment and professional personnel, can be carried out in clinical or family environment.

[0028] High sensitivity: using the sensitivity of FRET effect, low concentration of target enzyme can be detected.

[0029] Specificity: based on the specific recognition of enzymes, false positive results are reduced.

[0030] Stability: the transparent PEG hydrogel system provides a stable environment for detection, improving the reliability and reproducibility of the detection results.

[0031] In summary, the FRET diagnosis and treatment probe constructed by using CQDs / MOF composite system can provide a comprehensive solution in improving signal-to-noise ratio, enhancing light stability and realizing treatment function, providing a new path for the development of clinically convertible biological probes. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and the drawings.

[0033] Figure 1 The absorption spectrum and emission spectrum of the modified QD are shown in a and b respectively;

[0034] Figure 2 TEM observation results (a) and EDS energy spectrum analysis results (b) are shown in a and b respectively;

[0035] Figure 3 SEM image of the nanohydrogel probe;

[0036] Figure 4 Stress-strain curve of the nanohydrogel probe; wherein a is the mechanical strength detection of the hydrogel, and b is the transparency detection of the nanohydrogel probe;

[0037] Figure 5 Concentration response curve of the nanohydrogel probe; wherein a is the fluorescence image obtained under 365 nm excitation; the enzyme concentrations from right to left are 0 nM, 100 nM, 200 nM, 500 nM, 8000 nM, and 1000 nM; and b is the fluorescence emission spectrum obtained by a spectrometer;

[0038] Figure 6 Standard curve of the Pg pathogenic enzyme concentration;

[0039] Figure 7 Specific response curve of the nanohydrogel probe to different bacteria;

[0040] Figure 8 Influence of different concentrations of FRET probes on cell survival rate; wherein a is the Live / Dead staining image of the cells after treatment with different concentrations of FRET probes (green for living cells, red / black for dead cells; scale bar 50 μm); and b is the cell survival rate under the corresponding conditions;

[0041] Figure 9 Representative curve of ROS generation under light conditions;

[0042] Figure 10 In vitro antibacterial effect diagram; wherein a is the number of colonies corresponding to different treatment groups, and b is the OD value of the amount of biofilm corresponding to different treatment groups. DETAILED DESCRIPTION

[0043] The various illustrative embodiments of the present application will now be described in detail in connection with the following figures. This description is made for the purpose of illustrating the general principles of the present application and is not meant to limit the present application in its application to such principles. Further, the description of the illustrative embodiments is intended to convey the scope of the present application and is not meant to limit the application to the embodiments described.

[0044] It should be understood that the terms used herein are merely descriptive, but are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within the range of the intermediate values, and any other stated value or intermediate value within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0045] Unless otherwise indicated, all technical and scientific terms have the same meaning as those one of ordinary skill in the art of the disclosure would understand. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All documents mentioned herein are incorporated by reference to disclose and describe in further detail the methods and / or materials associated with the documents. In the case of conflict between the content of the specification and that of any document incorporated by reference, the content of the specification controls.

[0046] Many modifications and variations of the present disclosure described in the detailed description of the specification can be made without departing from the scope or spirit of the present disclosure, which will be apparent to those skilled in the art. Other implementations of the disclosure will be apparent to those skilled in the art from the specification. The specification and examples of the disclosure are merely illustrative.

[0047] As used herein, "comprise", "comprising", "have", "having", "include", "including", "contain", "containing", and the like, are open-ended terms that are intended to mean including, but not limited to.

[0048] Example 1 Preparation of FRET probe and nano MOF with photodynamic effect

[0049] This example aims to prepare a FRET probe for use in a diagnosis and treatment integrated system, and to elaborate on the synthesis of a nano metal organic framework (MOF) with intrinsic photodynamic effect and its functionalization.

[0050] 1) Synthesis and surface carboxylation modification of carbon quantum dots (CQDs)

[0051] Carbon quantum dots (CQDs) were prepared by a hydrothermal method: 1.0 g of citric acid was dissolved in 10 mL of deionized water and transferred to a polytetrafluoroethylene reaction kettle, and reacted at 180°C for 4 h. After cooling, the CQDs solution was dialyzed for 24 h through a 10 kDa dialysis bag. Subsequently, surface carboxylation was performed: 10 mL (5 mg·mL -1 ) of CQDs was taken, 50 mg of mercaptopropionic acid was added, the pH was adjusted to 8-10, and stirring was carried out at room temperature for 12 h to complete the ligand exchange. The free ligand was removed by dialysis again to obtain CQDs with a surface rich in carboxyl groups. The modified CQDs were observed by transmission electron microscopy (TEM) to have a circular nanomorphology with an average diameter of 5 nm. The optical properties were characterized by a fluorescence spectrometer, and the absorption and emission spectra are shown in Figure a-b. The figure shows that the CQDs have a clear absorption peak at about 340-360 nm, and the absorption rapidly decays at >400 nm; under 365 nm excitation, a strong single emission peak appears at about 410-430 nm, showing a large Stokes shift, which is beneficial to reducing self-absorption and improving the signal-to-noise ratio; the carboxylation treatment does not significantly change the optical properties. Figure 1

[0052] ​2) Preparation of Ag-modified MOF

[0053] 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 DMF in a 250 mL round-bottom flask and stirred at 90 °C for 5 h at 300 r / min. After the reaction was completed, the MOF nanoparticles were collected by centrifugation (15000 r / min, 30 min) and then washed with fresh DMF three times.

[0054] To load silver nanoparticles onto the surface of the MOF, the present application adopts a reduction method. First, 50 mg of the prepared MOF was uniformly dispersed in 50 mL of water by ultrasonic treatment for 20 min. Next, 150 μL of 0.1 M AgNO3 was added, ultrasonic mixing was performed for 20 min, and then the sample was moved to a magnetic stirring device and stirred at a speed of 800 rpm. During stirring, a freshly prepared NaBH4 solution (0.1 M, 3.75 mL) was quickly added, and the stirring speed was maintained at 800 rpm for 1 h. The sample was centrifuged at 6000 rpm and washed three times.

[0055] It can be seen from TEM observation that Ag nanoparticles are deposited on the MOF framework, as shown in FIG. 8a; EDS spectrum analysis further confirms that the nanoparticles contain Ag elements, indicating that Ag NPs are successfully loaded on the MOF, as shown in FIG. 8b. Figure 2 Figure 2

[0056] 3) Synthesis of peptide chain and construction of FRET probe

[0057] A polypeptide linker containing a sequence that can be recognized and cleaved by the target enzyme was prepared. A peptide chain recognizing and cleaving the sequence of arginine-specific cysteine protease (Rgp) was synthesized: GGFLVRGK (SEQ ID NO. 1).

[0058] The peptide chain contains a specific enzyme cleavage site, and the two ends are chemically modified: one end introduces an amino group to couple with the carboxyl group on the surface of CQDs; the other end introduces a thiol group to couple with the silver nanoparticles on the surface of the nano-MOF.

[0059] The modified CQDs, the modified peptide chain, and the modified MOF are connected in an optimized molar ratio of 10:10:1 to form a FRET probe with a CQDs-peptide chain-MOF ternary structure. The connection mode is to first couple the carboxylated quantum dots with the amino end of the polypeptide chain through an amino coupling reaction, and then couple the polypeptide chain thiol with the pre-set binding site (silver-sulfur bond site) on the surface of the MOF.

[0060] Preparation and encapsulation of nanohydrogel probe​​

[0061] This example aims to elaborate the preparation method of the optimized hydrogel carrier, as well as the encapsulation of FRET probe and nano MOF with photodynamic effect in the hydrogel.

[0062] 1) Preparation of PEG hydrogel solution

[0063] An appropriate amount of maleimide-terminated polyethylene glycol (PEG-MAL, 10 kDa) and thiol-terminated polyethylene glycol (PEG-SH, 3 kDa) were weighed and dissolved in HEPES buffer (pH 7.2) respectively to prepare a solution with a certain concentration (7%, w / v). When preparing the solution, the molar ratio of maleimide groups to thiol groups (1:1) should be ensured to facilitate efficient crosslinking.

[0064] 2) Encapsulation of FRET probe and hydrogel formation

[0065] 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, ensuring uniform dispersion of the nanoparticles in the solution. Subsequently, the two PEG solutions were mixed and thoroughly mixed. The mixed solution containing nanoparticles was quickly injected into a pre-designed mold. At room temperature, rapid crosslinking occurred between PEG-MAL and PEG-SH through Michael addition reaction, causing the formation and solidification of the hydrogel network, thereby effectively encapsulating the nano-diagnostic and therapeutic particles. The solidification time of the hydrogel can be completed within 5 min. The solidified hydrogel was removed from the mold and washed thoroughly with HEPES solution to remove unreacted substances, thereby obtaining the nano-hydrogel probe.

[0066] The prepared nano-hydrogel probe was characterized for performance characteristics: the internal pore structure of the hydrogel was observed by scanning electron microscopy (SEM), and the results are shown in Figure 3 , which shows that the average pore size of the nano-hydrogel probe is 500 nm, which can ensure its diffusion of enzymes, substrates and singlet oxygen. The mechanical strength of the hydrogel was tested by a universal testing machine, and the results are shown in Figure 4 , which shows that the stress increases nearly linearly with strain during stretching, and breaks at about 240% strain, with a limit stress of about 0.9 MPa; indicating that it has high ductility and good mechanical strength, with uniform network structure and stable crosslinking. At the same time, the transparency of the nano-hydrogel probe was observed by ultraviolet-visible spectrophotometer Figure 4 b), and the detection results found that the transmittance of the nano-hydrogel probe reached more than 97% in the wavelength range of 400-800 nm, which can ensure the reading of fluorescence signal during detection and the penetration of light during photodynamic therapy.

[0067] Example 3 Detection and photodynamic therapy function verification of nanohydrogel probe

[0068] This example aims to verify the function and effect of the nanohydrogel probe in detection and photodynamic therapy by taking the pathogenic enzyme of Porphyromonas gingivalis-gingipain as an example.

[0069] 1. Detection performance verification

[0070] 1) Concentration response and sensitivity

[0071] Different concentrations (0-1000 nM) of purified Porphyromonas gingivalis pathogenic enzyme solution were used as the sample to be tested and added dropwise to the nanoprobes containing FRET probes prepared by the present application. After incubation for a specific time of 10 min, the fluorescence signal of the nanohydroprobe was measured using a fluorescence spectrometer. The increase in QD (blue light region) fluorescence intensity and the decrease in MOF (red light region) fluorescence intensity were recorded, and a standard curve of fluorescence signal ratio (e.g. blue / red intensity ratio) versus Pg pathogenic enzyme concentration was constructed to determine the detection sensitivity, limit of detection (LOD) and linear range of the nanohydrogel probe.

[0072] The results are shown in a-b of Figure 5 , which shows the fluorescence signal changes of the nanohydrogel probe of the present application under different concentrations of Porphyromonas gingivalis pathogenic enzyme. With the increase of the concentration of pathogenic enzyme, the red light region signal representing the fluorescence emission intensity of MOF showed a downward trend, while the blue light region signal representing the fluorescence emission intensity of carbon quantum dots increased significantly. This result strongly verifies that the nanohydrogel probe of the present application has excellent concentration-dependent response and high sensitivity to the target pathogenic enzyme, and further suggests that it has the potential to realize ratio fluorescence detection, thereby improving the accuracy and anti-interference ability of detection. According to the standard curve (as shown in Figure 6 ) constructed, the LOD of the nanohydrogel probe is 5 nM, which can meet the clinical needs.

[0073] 2) Detection specificity

[0074] The nanoprobes prepared by the present application were exposed to the same concentration (10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8The system was tested in cultures or lysates of *Porphyromonas gingivalis* (per / mL) and other common oral bacteria, including *Fusobacterium nucleatum*, *Streptococcus oralis*, and *T. forsythia*. Fluorescence signals were measured after 10 min of incubation. The specific response of this system to the Pg pathogenic enzyme was verified, and its cross-reactivity with non-target bacteria was evaluated.

[0075] The experimental results are shown in Figure 7 , Figure 7 The study showed that at a concentration of 10... 4 Up to 10 5 Within the per mL range, the fluorescence signal at 420 nm showed a sharp increase and reached saturation at higher concentrations. However, for controls such as *Fusobacterium nucleatum*, *Streptococcus oralis*, and *T. forsythia*, the detection signals remained at low levels even at similar or higher bacterial concentrations. This fully demonstrates that the nanohydrogel probe prepared in this invention has excellent specific recognition ability for *Porphyromonas gingivalis*, effectively distinguishing the target pathogen from other common bacteria in the oral cavity, providing a reliable basis for accurate diagnosis.

[0076] 2. Biosafety testing

[0077] 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.

[0078] Experimental results are as follows Figure 8 As shown in Figure 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.

[0079] 3. Validation of photodynamic therapy function

[0080] 1) Singlet oxygen generation ability

[0081] The nanohydrogel probe encapsulating MOF was exposed to light (660 nm, light power density 30 mW / cm 2 ) for 20 min. The amount of singlet oxygen generated in the solution was detected by a specific singlet oxygen fluorescent probe to prove its photodynamic effect.

[0082] The experimental results are shown in a-b of Figure 9 The chart shows that the probe produces reactive oxygen species (ROS) under light conditions. Each light-on event produces a set of obvious positive and negative peaks, and the signal returns to near baseline after the light is turned off, indicating that ROS generation has light-triggered, reversible, and repeatable characteristics.

[0083] 2) In vitro antibacterial effect

[0084] A P. gingivalis biofilm model or a suspension bacterial solution was constructed in vitro. The nanohydrogel probe containing MOF was placed therein and subjected to light treatment (light parameters: wavelength 660 nm, light power density 10-50 mW / cm 2 ) for 20 min, and PBS buffer and nanohydrogel probe dark treatment were used as two controls. The survival rate of bacteria or the inhibition / clearance rate of biofilm was evaluated by colony counting and biofilm amount (crystal violet staining).

[0085] The experimental results are shown in a-b of Figure 10 The experimental results confirm the photodynamic antibacterial effect of MOF after encapsulation in the nanohydrogel probe, and the nanohydrogel probe prepared by the application can effectively reduce the number of P. gingivalis colonies (a in Figure 10 ), and has a high inhibition / clearance rate of biofilm (b in Figure 10 ), that is, the nanohydrogel probe has excellent in vitro antibacterial effect.

[0086] The above-described embodiments are only descriptions of preferred modes of the application and do not limit the scope of the application. Without departing from the design spirit of the application, various modifications and improvements to the technical solutions of the application made by those of ordinary skill in the art should fall within the protection scope of the claims of the application.

Claims

1. A method for preparing a MOF-CQD enzyme-sensitive FRET hydrogel diagnosis and treatment probe, characterized in that, The preparation method comprises the following steps: CQDs synthesis and surface carboxyl modification: CQDs are synthesized by high-temperature thermal method, and the surface of the CQDs is modified by a carboxyl-containing ligand to obtain modified CQDs; Preparation of Ag-modified MOF: H2TCPP, ZrOCl2.8H2O and benzoic acid are dissolved in DMF, stirring, centrifugation, washing to obtain MOF nanoparticles; the MOF is dispersed into water, AgNO3 is added, ultrasonic mixing, stirring and adding NaBH4 solution, centrifugation, washing to obtain Ag-modified MOF; Construction of FRET probe: a peptide chain for recognizing and cutting enzyme sequence is synthesized; the two ends of the peptide chain are chemically modified, one end is introduced with amino group and the other end is introduced with mercapto group; the modified CQDs, the peptide chain and the MOF obtained above are connected to form a FRET probe; Preparation of nano-hydrogel probe: the obtained FRET probe is added into a maleimide-terminated polyethylene glycol solution or a thiol-terminated polyethylene glycol solution at a concentration of 50-150 μg / mL, then the two polyethylene glycol solutions are mixed, and the mixture is injected into a mold for solidification to obtain a nano-hydrogel probe.

2. The production method according to claim 1, characterized by, The sequence of the peptide chain is shown as SEQ ID NO.

1.

3. The preparation method according to claim 1, characterized in that, The 50-150 mg H2TCPP with a concentration of 0.1-0.15 mM, 100-500 mg ZrOCl2.8H2O with a concentration of 0.93-1.0 mM and 2.6-3.0 g benzoic acid with a concentration of 20-25 mM are dissolved in 50-150 mL DMF, stirring at a speed of 100-500 r / min at 80-100 ℃ for 3-8 h, centrifugation at 10,000-20,000 r / min for 10-50 min, and washing to obtain MOF nanoparticles.

4. The production method according to claim 3, characterized by, The 100 mg H2TCPP with a concentration of 0.13 mM, 300 mg ZrOCl2.8H2O with a concentration of 0.93 mM and 2.8 g benzoic acid with a concentration of 23 mM are dissolved in 100 mL DMF, stirring at a speed of 300 r / min at 90 ℃ for 5 h, centrifugation at 15,000 r / min for 30 min, and washing to obtain MOF nanoparticles.

5. The preparation method according to claim 3, characterized in that, The prepared MOF is dispersed into 10-90 mL water for 10-30 min, 100-200 μL AgNO3 with a concentration of 0.05-0.15 M is added, ultrasonic mixing is performed for 10-30 min, stirring is performed at 600-1,000 rpm and 3.5-4.0 mL NaBH4 solution with a concentration of 0.05-0.15 M is added, stirring is performed for 0.5-1.5 h, centrifugation and washing are performed to obtain Ag-modified MOF.

6. The production method according to claim 5, wherein The prepared MOF is dispersed into 50 mL water for 20 min, 150 μL AgNO3 with a concentration of 0.1 M is added, ultrasonic mixing is performed for 20 min, stirring is performed at 800 rpm and 3.75 mL NaBH4 solution with a concentration of 0.1 M is added, stirring is performed for 1 h, centrifugation and washing are performed to obtain Ag-modified MOF.

7. The preparation method according to claim 1, characterized in that, The modified CQDs, peptide chains and MOF are connected in a molar ratio of 10:10:1 to obtain a FRET probe; The concentration of the maleimide-terminated polyethylene glycol solution and the thiol-terminated polyethylene glycol solution is 7%; the FRET probe is added to the maleimide-terminated polyethylene glycol solution or the thiol-terminated polyethylene glycol solution at a concentration of 100 μg / mL.

8. The hydrogel diagnosis and treatment probe prepared by the preparation method according to any one of claims 1-7.

9. A product for detecting Porphyromonas gingivalis, characterized by, The product comprises the hydrogel diagnosis and treatment probe according to claim 8.

10. A product for inhibiting the growth of Porphyromonas gingivalis, characterized by, The product comprises the hydrogel diagnosis and treatment probe according to claim 8.

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