A curcumin-based carbon dot nanocatalytic cascade system and its application in targeted anti-cariogenic bacteria

Abstract

This invention discloses a curcumin-based carbon dot nanozyme cascade catalytic system and its application in targeted anti-cariogenic bacteria, belonging to the field of biomedical technology. The catalytic system is prepared by mixing curcumin-based carbon dot nanozymes (Cur@Fe,Mn / CDs) and glucose oxidase (GOx), followed by ultrasonic incubation. The curcumin-based carbon dot nanozyme cascade catalytic system exhibits near-infrared enhanced peroxidase-like activity and excellent photothermal properties. It utilizes the hydrogen peroxide generated from GOx oxidizing glucose as a substrate for the peroxidase-mimicking enzyme Cur@Fe,Mn / CDs, generating highly toxic hydroxyl radicals under acidic conditions. By leveraging the positively charged surface of the nanozyme and the precise positioning of GOx on the biofilm, it can effectively target cariogenic bacteria. S. mutans and S. sanguinis It targets and inhibits cariogenic bacteria while protecting normal oral cavity. S. oralis With virtually no impact, this invention provides a new approach to the treatment of dental caries.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a curcumin-based carbon dot nanozyme cascade catalytic system and its application in near-infrared light-enhanced targeted anti-cariogenic bacteria. Background Technology

[0002] Dental caries, or dental decay, is a disease that is stable in the enamel or hard tissues of the teeth. It is one of the most common chronic infectious diseases worldwide, with a prevalence of 35% across all age groups. The formation of caries in the hard tissues of human teeth is directly related to plaque biofilm in the oral environment. Notably, cariogenic bacteria such as Streptococcus mutans (Streptococcus mutans) Streptococcus mutans Dental caries are caused by the production of acid, and biofilms, consisting of well-structured microorganisms embedded in an extracellular polymeric substance (EPS) matrix, form a structurally stable and mechanically rigid protective barrier that can cause persistent human infections. Anaerobic plaque biofilms also contain high concentrations of H2O2. The primary goal of caries prevention is to remove stubborn plaque biofilms and reduce tooth demineralization. However, while mechanical decontamination in clinical practice can effectively remove plaque biofilms, it is insufficient to clean inaccessible areas and often damages the tooth surface. Many synthetic agents, such as chlorhexidine, quaternary ammonium salts, fluoride, and high concentrations of H2O2 (3%), have been widely used to remove bacteria from the oral cavity, disrupting the microbial community balance. Therefore, it is essential to explore an integrated platform for the specific capture, targeting, and removal of pathogens while minimizing damage to the normal microbiota and surrounding tissues.

[0003] Recently, research on peroxidase-like nanozymes based on metal-doped carbon dots (CDs) in the field of antibacterial applications has been increasing. Iron-doped carbon dots combine the catalytic and optical properties of metals with the size effect of quantum dots, effectively combating bacteria through synergistic effects with chemical kinetics, photothermal, and photodynamic pathways. Simultaneously, CDs possess high photothermal conversion capabilities. When exposed to near-infrared light (NIR), the surface electrons of CDs absorb light energy and undergo a transition from the ground state to an excited state, then return to the ground state, releasing energy as heat, generating a photothermal effect. This rapidly increasing temperature kills bacteria, i.e., photothermal therapy (PTT). Therefore, peroxidase-like nanozymes based on doped CDs offer the possibility of synergistic effects between photodynamic therapy (PDT) and PTT antibacterial mechanisms. The mechanism of action of PDT is the activation of photosensitizers under laser irradiation to generate large amounts of cytotoxic singlet oxygen (PTO). 1 O2) and superoxide radicals (O2) ·- ( ) to remove biofilm, while PTT uses photothermal agents (PTAs) that absorb near-infrared light to generate local heat therapy to remove biofilm. Summary of the Invention

[0004] Based on the problems raised in the background technology, this invention provides a curcumin-based carbon dot nanozyme cascade catalytic system and its application in the preparation of near-infrared light-enhanced targeted anti-cariogenic bacteria preparations. This invention prepares a multifunctional Fe,Mn-doped curcumin-based carbon dot integrated glucose oxidase nanozyme cascade catalytic system (Cur@Fe,Mn / CDs@GOx) via a one-step microwave digestion method. Cur@Fe,Mn / CDs@GOx exhibits near-infrared enhanced peroxidase-like activity and excellent photothermal properties. Utilizing the hydrogen peroxide (H2O2) generated by GOx oxidizing glucose as the substrate of the peroxidase-mimicking enzyme Cur@Fe,Mn / CDs, highly toxic hydroxyl radicals are generated under acidic conditions. Simultaneously, the positively charged surface of the nanozyme and the precise positioning of GOx on biofilms enable it to target bacteria including Streptococcus mutans (Streptococcus mutans). Streptococcus mutans ) and Streptococcus sanguinis ( Streptococcus sanguinis The photocatalytic cascade catalytic system significantly increased the production of reactive oxygen species (ROS), effectively eliminating cariogenic pathogens and biofilms. In vitro experiments showed that Cur@Fe,Mn / CDs@GOx exhibited excellent biocompatibility, showing good resistance to cariogenic bacteria at a concentration of 50 μg / mL. S. mutans and S. sanguinis The sterilization efficiency all exceeded 98%, and they effectively inhibited... S. mutans and S. sanguinis The formation of biofilms provides a novel cariogenic targeted antibacterial method, offering a new approach to the treatment of dental caries.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: 1. Dissolve 0.5-0.8g of curcumin in 1-2 mL of anhydrous ethanol, then add 0.3-0.5g of citric acid, 0.35-0.50g of FeCl3·6H2O, 0.35-0.50g of MnCl2·4H2O, and 30-50 mL of acetic acid aqueous solution with a weight-to-volume ratio of 1-2% (g:mL). After sonicating the mixture for 20-30 min, react it at 170-190℃ for 100-150 min using a microwave power of 1.0-1.2 kW. After cooling the reaction product, centrifuge it, filter the supernatant through a 0.22 mm filter membrane, and vacuum dry it to obtain curcumin-based carbon dot nanozyme Cur@Fe,Mn / CDs. 2. Mix curcumin-based carbon dot nanozymes Cur@Fe,Mn / CDs with glucose oxidase GOx in equal mass, sonicate for 60-90 min, and then incubate at 4℃ for 8-10 h to obtain the curcumin-based carbon dot nanozyme cascade catalytic system Cur@Fe,Mn / CDs@GOx. 3. Mix the curcumin-based carbon dot nanozyme cascade catalytic system Cur@Fe,Mn / CDs@GOx with glucose until homogeneous. When using, irradiate the mixture under near-infrared light at 808nm for 10-15 minutes to achieve targeted anti-cariogenic bacteria. The weight ratio of the curcumin-based carbon dot nanozyme cascade catalytic system Cur@Fe,Mn / CDs@GOx to glucose is 1:100-150. The cariogenic bacteria is Streptococcus mutans ( ) Streptococcus mutans ) and Streptococcus sanguinis ( Streptococcus sanguinis ) The power of the 808 nm near-infrared light is 0.50-1.0 W / cm². 2 .

[0006] The beneficial effects of this invention are: 1. This invention synthesizes Fe,Mn-doped curcumin-based carbon dots (Cur@Fe,Mn / CDs@GOx) nanozymes with positively charged surfaces and modified with glucose oxidase. The introduction of Fe and Mn enhances the peroxidase-like activity responsive to infrared light, while the introduction of curcumin gives it a positive surface charge, ultimately giving Cur@Fe,Mn / CDs excellent peroxidase-like activity and photothermal properties. Hydrogen peroxide (H2O2) generated by GOx oxidation of glucose is used as the substrate for the peroxidase-mimicking enzyme Cur@Fe,Mn / CDs, producing highly toxic hydroxyl radicals under acidic conditions. Simultaneously, the positively charged surface of the nanozyme and the precise positioning of GOx on biofilms enable it to target bacteria including Streptococcus mutans (Streptococcus mutans). S.mutans ) and Streptococcus sanguinis ( S.sanguinis It targets and inhibits bacteria in the oral cavity, while inhibiting normal oral streptococci ( S.oralis The near-infrared light significantly enhanced the peroxidase-like activity of Cur@Fe,Mn / CDs, with almost no effect. Furthermore, the near-infrared light significantly enhanced the activity of Cur@Fe,Mn / CDs at a concentration of 50 μg / mL, at 1.0 W / cm². 2 After irradiation with 808nm NIR for 10 minutes, the effect on S. mutans and S. sanguinis The sterilization efficiency all exceeded 98%, and they effectively inhibited... S. mutans and S. sanguinis Biofilm formation, while affecting normal oral streptococci ( S. oralis There was no effect, which is attributed to the substrate channel effect and proximity effect of Cur@Fe,Mn / CDs@GOx. The excellent peroxidase-like activity generated reactive oxygen species and NIR promoted the electron transport mechanism in the bacterial membrane, triggering the electron transport process, which led to electrons entering the bacterial nucleus and ultimately causing bacterial death. 2. In vitro experiments show that Cur@Fe,Mn / CDs@GOx has excellent biocompatibility and low toxicity. This four-modal synergistic system of "targeted penetration-catalysis-depletion-photothermal" provides a new model for developing targeted therapy for caries. Attached Figure Description

[0007] Figure 1 The image shows a TEM image of Cur@Fe,Mn / CDs prepared in Example 1 of this invention, where a is 20 nm and b is 2 nm. Figure 2 The FT-IR image of Cur@Fe,Mn / CDs prepared in Example 1 of this invention; Figure 3 The high-resolution XPS spectra of Fe 2p(a) and Mn 2p(b) of Cur@Fe,Mn / CDs prepared in Example 1 of this invention are shown. Figure 4 Zeta potential diagram of the material and / or cariogenic bacteria prepared in Example 1 of the present invention; Figure 5 The temperature rise curves for different concentrations of Cur@Fe,Mn / CDs@GOx in Example 1 are shown. Figure 6 The temperature-time graph (a) and photothermal conversion rate curve (b) of Cur@Fe,Mn / CDs@GOx in Example 1 of the present invention are shown. Figure 7 The UV-Vis absorption spectra of Cur@Fe,Mn / CDs and Cur@Fe,Mn / CDs+NIR in Example 1 of this invention; Figure 8 The UV-Vis absorption spectra of Cur@Fe,Mn / CDs@GOx+Glu and Cur@Fe,Mn / CDs@Gox+Glu +NIR in Example 1 of this invention; Figure 9 The Michaelis-Menten equation for Cur@Fe,Mn / CDs@GOx in Example 1 is as follows: with a fixed H2O2 concentration but varying TMB concentration. Figure 10 The Michaelis-Menten equation for Cur@Fe,Mn / CDs@GOx in Example 1 is given by varying the H2O2 concentration while keeping the TMB concentration constant. Figure 11 The Michaelis-Menten equation for Cur@Fe,Mn / CDs@GOx+NIR in Example 1 is given by changing the TMB concentration while keeping the H2O2 concentration constant. Figure 12 The Michaelis-Menten equation for Cur@Fe,Mn / CDs@GOx+NIR in Example 1 is given by changing the H2O2 concentration while keeping the TMB concentration constant. Figure 13The effects of different concentrations of Cur@Fe,Mn / CDs@GOx on the survival rates of Hep G2 cells (a) and HUVECs cells (b) at 24 h and 48 h were investigated. Figure 14 In Example 1, (a) the effect of different experimental groups on the antibacterial properties of different bacteria under 808nm irradiation and no irradiation; and (b) the effect of different concentrations of Cur@Fe,Mn / CDs@GOx on the antibacterial properties of different bacteria under 808nm irradiation and no irradiation. Figure 15 In Example 1, SEM images of different bacteria were processed using Cur@Fe,Mn / CDs@GOx+Glu and Cur@Fe,Mn / CDs@Gox+Glu+NIR. Figure 16 In Example 1, ROS images of different bacteria were processed using Cur@Fe,Mn / CDs@GOx+Glu and Cur@Fe,Mn / CDs@Gox+Glu +NIR. Figure 17 In Example 1, Cur@Fe,Mn / CDs@GOx+Glu and Cur@Fe,Mn / CDs@Gox+Glu+NIR inhibition were used. S. mutans The result of biofilms; Figure 18 For the Cur@Fe,Mn / CDs@GOx+Glu and Cur@Fe,Mn / CDs@Gox+Glu+NIR inhibition in Example 1 S. sanguinis The result of biofilms; Figure 19 For the Cur@Fe,Mn / CDs@GOx+Glu and Cur@Fe,Mn / CDs@Gox+Glu+NIR inhibition in Example 1 S. oralis The result of biofilms; Figure 20 In Example 1, Cur@Fe,Mn / CDs@GOx+Glu and Cur@Fe,Mn / CDs@Gox+Glu+NIR inhibition were used. S. mutans , S. sanguinis and S. oralis 3D test results of biofilm. Detailed Implementation

[0008] The present invention will be further described in detail below with reference to the embodiments. However, the content described should not be regarded as a limitation of the present invention. Unless otherwise specified, the methods in this embodiment are operated in accordance with conventional methods, and the reagents used are conventional reagents or reagents prepared in accordance with conventional methods unless otherwise specified.

[0009] Example 1: Preparation, characterization, and properties of Cur@Fe,Mn / CDs@GOx nanomaterials 1. Preparation of Cur@Fe,Mn / CDs: 0.5g curcumin was dissolved in 1mL anhydrous ethanol, and 0.3g citric acid, 0.35g FeCl3·6H2O, 0.35g MnCl2·4H2O, and 30mL of acetic acid aqueous solution with a weight / volume ratio of 1% (g:mL) were added. The mixture was sonicated for 30min, and then reacted at 180℃ for 120min using a microwave power of 1.0kW. After cooling, the reaction product was centrifuged at 8000r / min for 10min. The supernatant was filtered through a 0.22mm filter membrane and vacuum dried to obtain curcumin-based carbon dot nanozymes Cur@Fe,Mn / CDs. Transmission electron microscopy (TEM) verified that the Cur@Fe,Mn / CDs nanoparticles all exhibited a monodisperse spherical structure with a narrow size distribution (the average particle size of 100 random nanoparticles was 2.66nm). Figure 1 (a); Meanwhile, high-resolution TEM (HRTEM) images ( Figure 1 Image b) shows clear lattice fringes with a lattice spacing of 0.22 nm, belonging to sp. 2 - The (100) characteristic surface of graphitic carbon; then, the distribution of surface functional groups was analyzed by FT-IR, such as Figure 2 As shown, it is located at 3431cm. -1 1712cm -1 1604cm -1 and 1422cm -1 The spectral bands are attributed to the stretching vibrations of the -OH, C=O, C=C, and CO groups, respectively, at 678 cm⁻¹. -1 and 571cm -1 They are respectively attributed to the Mn-O and Fe-O stretching vibrations; Figure 3 In the high-resolution XPS spectrum of Fe 2p in α, the two pairs of characteristic peaks are attributed to Fe. 2+ 2p 3 / 2 (710.1eV), Fe 3+ 2p 3 / 2 (715.0eV), Fe 2+ 2p 1 / 2 (724.2eV), Fe 3+ 2p 1 / 2 (732.2 eV), indicating the presence of Fe. 2+ and Fe 3+ Fe 2+ The presence of CDs indicates that they have partial iron reduction properties; Figure 3 In the high-resolution spectrum of Mn 2p in b, 634.7 eV and 652.2 eV are attributed to Mn, respectively.4+ 2p 3 / 2 and Mn 3+ 2p 1 / 2 This indicates the existence of Mn 4+ and Mn 3+ Coexistence; Zeta potential analysis is used to test the surface charge of materials and cariogenic bacteria. Figure 4 The results showed that the surface of Cur@Fe,Mn / CDs was positively charged, while the surface of cariogenic bacteria was negatively charged, and the surface of Cur@Fe,Mn / CDs+ cariogenic bacteria was generally negatively charged.

[0010] 2. Preparation of Cur@Fe,Mn / CDs@GOx: Mix 1 mL of 400 μg / mL Cur@Fe,Mn / CDs with 1 mL of 400 μg / mL glucose oxidase GOx, sonicate for 60 min, and then incubate the mixture at 4 °C for 8 h to obtain Cur@Fe,Mn / CDs@GOx.

[0011] 3. Evaluation of the photothermal performance of Cur@Fe,Mn / CDs@GOx: To assess the NIR-triggered photothermal performance of the Cur@Fe,Mn / CDs@GOx nanozyme, the temperature change of different concentrations of Cur@Fe,Mn / CDs@GOx under 808 nm infrared irradiation was measured. The photothermal curves confirmed that the temperature increase was positively correlated with the concentration of Cur@Fe,Mn / CDs@GOx. Figure 5 Under irradiation with light at a wavelength of 808 nm, the temperature of a 200 µg / mL Cur@Fe,Mn / CDs@GOx solution rose to 53.3 °C. This temperature range effectively induced damage to the bacterial membrane structure. In contrast, the temperature increase of pure water under 808 nm infrared irradiation was only 30.5 °C. These results demonstrate that Cur@Fe,Mn / CDs@GOx possesses the ability to convert NIR light energy into heat energy, providing a foundation for further research on the photothermal properties of Cur@Fe,Mn / CDs@GOx.

[0012] In addition, the photothermal conversion efficiency of 150 μg / mL Cur@Fe,Mn / CDs@GOx was calculated using the following formula ( η T ): ; in T max It is the equilibrium temperature of the sample solution. T surr Corresponding to the ambient temperature of the experiment, Q dis This is the heat loss caused by the container absorbing light, among which Q dis = (5.4×10-4 ), where I represents the power density of the 808nm laser (1.0W / cm²). 2 The absorbance of the sample solution at 808 nm is denoted as . A λ In order to determine hs The value of is obtained using the following formula: ; in m Indicates the mass of the sample solution. C water Corresponding to the heat capacity of water (4.2 J·kg) −1 ·K −1 ), τ s This is the system's time constant. Therefore, the photothermal conversion efficiency of Cur@Fe,Mn / CDs@GOx is 35.23% ( Figure 6 It exhibits a relatively high photothermal conversion efficiency.

[0013] 3. Evaluation of Cur@Fe,Mn / CDs-like peroxidase nanozyme activity: Using TMB as the catalytic substrate, its peroxidase-like (POD-like) activity was evaluated. In a 5 mL stoppered colorimetric tube, 100 μL of 5 mmol / L TMB, 100 μL of 50 mmol / L H₂O₂, 50 μL of 1 mg / mL Cur@Fe,Mn / CDs, and 2 mL of 0.1 mol / L HAc-NaAc buffer solution (pH 4.0) were added and thoroughly mixed. The volume was then adjusted to 3 mL with deionized water. After reacting for 10 min, the absorbance was measured. Simultaneously, the experiment was conducted at 1.0 W / cm² under the same conditions. 2 The enzyme activity after irradiation at 808 nm for 10 min was compared, and the absorbance was measured at a wavelength of 654 nm; the results are as follows. Figure 7 When Cur@Fe,Mn / CDs and H2O2 were present in the system, TMB underwent a significant oxidation reaction. This experimental phenomenon fully demonstrates that Cur@Fe,Mn / CDs possess POD-like catalytic activity, capable of mimicking the function of natural peroxidase, and catalyzing the oxidation of TMB by H2O2 to produce a blue product. Furthermore, when we applied 1.0 W / cm² to the system... 2 After irradiation at 808 nm for 10 min, the POD-like activity of Cur@Fe,Mn / CDs was significantly enhanced, with an increase of nearly 2.9 times. Cur@Fe,Mn / CDs exhibited the characteristic of near-infrared light enhancing peroxidase-like activity.

[0014] 4. Evaluation of the cascade catalytic system of Cur@Fe,Mn / CDs@GOx: Add 100 μL of 5 mmol / L TMB, 100 μL of 4 mmol / L glucose (Glu), 50 μL of 1 mg / mL Cur@Fe,Mn / CDs@Gox, and 2 mL of 0.1 mol / L HAc-NaAc buffer solution (pH 4.0) to a 5 mL stoppered colorimetric tube. Mix thoroughly, bring the volume to 3 mL with deionized water, and react for 10 min. Measure the absorbance at 654 nm. Simultaneously, experimental groups were set up: Glu+GOx+TMB, TMB+Cur@Fe,Mn / CDs, Glu+TMB+Cur@Fe,Mn / CDs, and Glu+TMB+Cur@Fe,Mn / CDs@GOx+NIR. The results are as follows: Figure 8 After NIR irradiation for 10 min, the POD-like activity of Cur@Fe,Mn / CDs@Gox was significantly enhanced.

[0015] The experiment also included the determination of Michaelis catalytic kinetic parameters. Figure 9-12 (and Table 1), Michaelis constants of Cur@Fe,Mn / CDs@GOx for substrates TMB and H2O2. K m The reaction rate constants are 0.49 × 10⁻⁶ mM and 0.36 mM, respectively. -7 M / s and 0.23×10 -7 M / s, after infrared light irradiation K m The reaction rates are 0.09 mM and 0.28 mM, with a rate constant of 3.07 × 10⁻⁶. -7 M / s and 2.73×10 -7 M / s indicates that infrared light irradiation increased the affinity and reaction rate of the Cur@Fe,Mn / CDs@GOx nanozyme with the substrate; Table 1 Michaelis catalytic kinetic parameters ; 5. Cytotoxicity test The cytotoxicity of nanozymes was detected using a CCK-8 cell viability assay kit. Specifically, human umbilical vein endothelial cells (HUVECs) and hepatocellular carcinoma cells (HepG2, from Beina Chuanglian Biotechnology Co., Ltd.) were seeded in 96-well plates and cultured for 24 h. After incubation with different concentrations of Cur@Fe,Mn / CDs@GOx for 24 h and 48 h, respectively, the cells were washed with PBS. CCK-8 solution was added to each well to a concentration of 10%, and the cells were incubated at 37 °C. The absorbance was measured at 450 nm. CCK-8 analysis (…) Figure 13 The results showed that Cur@Fe,Mn / CDs@GOx were not toxic to cells.

[0016] 6. Cur@Fe,Mn / CDs@GOx antibacterial test Streptococcus mutans obtained from Beina Chuanglian Biotechnology Co., Ltd. S. mutans BNCC336931), oral streptococci ( S. oralis BNCC354691) and Streptococcus sanguinis ( S. sanguinis (BNCC354356) was the experimental bacteria. S. mutans The cultures were performed using BHI medium at 37°C and 5% CO2. S.oralis and S. sanguinis The bacteria were cultured on Columbia Blood Agar (CA-B) medium at 37°C and 5% CO2. The antibacterial activity of Cur@Fe,Mn / CDs@GOx was determined by counting CFUs using the plate count method. The experiment was divided into the following groups: blank control group, positive control group (chlorhexidine hydrochloride), Glu+GOx group, Cur@Fe,Mn / CDs@GOx+Glu group, and Cur@Fe,Mn / CDs@GOx+Glu+NIR group, where the Glu concentration was 5 mmol / L and the chlorhexidine hydrochloride concentration was 50 μg / mL. The cultured bacteria were added to phosphate-buffered saline (PBS) as a blank control group, while the other groups were mixed with nanozymes or other reagents and subjected to a 1.0 W / cm² buffer solution. 2 The bacterial suspension, after being irradiated with 808nm NIR for 10 min or without infrared irradiation, was diluted (100 μL) and evenly spread onto the culture medium after incubation at 37℃ for 60 min. The culture was then incubated at 37℃ for 24 h, and the colony count was calculated to determine the antibacterial performance. Figure 14 (a)

[0017] The antibacterial properties of different concentrations of Cur@Fe,Mn / CDs@GOx against different bacteria were compared under 808nm irradiation and without irradiation. Figure 14 The results showed that the blank control group had almost no antibacterial activity. When Cur@Fe,Mn / CDs@GOx concentration was 50 μg / mL and Glu was 5 mmol / L, under 808 nm irradiation for 10 min, the antibacterial activity was significantly reduced. S.mutans and S.sanguinis It exhibits excellent antibacterial effects, with a near 100% kill rate against all bacteria, and is particularly effective against oral streptococci (Streptococcus spp.). S.oralis It has almost no impact.

[0018] 7. SEM observation of bacterial morphology SEM ( Figure 15 Characterization showed that the control group retained intact cell morphology; however, in the presence of Cur@Fe,Mn / CDs@GOx+Glu, S.mutans and S.sanguinis The cell membrane exhibits wrinkling and shrinkage. S.oralis There was no change, but after Cur@Fe,Mn / CDs@GOx+Glu+NIR treatment, S.mutans and S.sanguinis The cells were completely destroyed. S.oralis There was no impact whatsoever.

[0019] 8. Monitoring of intracellular ROS in bacteria Finally, intracellular reactive oxygen species (ROS) were measured using laser confocal scanning microscopy. Nanozymes, Glu, bacteria, and DCFH-DA were co-incubated to verify ROS generation; the more ROS generated, the stronger the observed fluorescence intensity. Figure 16 As shown, the Cur@Fe,Mn / CDs@GOx+Glu+NIR group in S.mutans and S.sanguinis The presence of a significant green fluorescence signal indicates that ROS production was highest in both groups. S.oralis The fluorescence intensity of the group was almost the same as that of the control group. This result further verifies that the cascade reaction of Cur@Fe,Mn / CDs@GOx can effectively generate more toxic ROS, thereby achieving targeted antibacterial function.

[0020] 9. Inhibition of biomembranes To evaluate the anti-biofilm properties of Cur@Fe,Mn / CDs@GOx, we investigated its anti-biofilm ability using crystal violet (CV) staining. Bacterial biofilms were added to 24-well plates, and Glu, nanozymes, or other reagents were added according to the grouping steps in step 6. Then, 200 μL of methanol was added to each well for fixation, and after 30 min, excess methanol was aspirated, and the plates were air-dried under natural conditions. Next, 200 μL of 1% CV staining agent was added to stain the biofilm. After 30 min of staining, excess staining agent was rinsed off with sterile PBS buffer. 200 μL of 33% glacial acetic acid solution was added to the rinsed biofilm, and incubation was performed for 30 min to dissolve the fixed biofilm. Finally, the OD value at 590 nm was measured, and the biofilm disruption strength of the nanocomposite was evaluated by the absorbance.

[0021] The results are as follows Figure 17-19 In the control group, biofilm formation was clearly observed. Cur@Fe,Mn / CDs@Gox (100 μg / mL) + Glu (5 mmol / L) + NIR significantly inhibited biofilm formation after 48 h of incubation. S.mutans and S.sanguinis Biofilm formation was characterized by minimum biofilm survival rates of 6.20% and 15.04%, while... S.oralis The inhibition of the biofilm was relatively weak, at 67.06%.

[0022] Visualization 3D biofilm test: To further evaluate the effects of Cur@Fe,Mn / CDs@Gox on... S.mutans , S.sanguinis and S.oralis To mitigate the destructive properties of the biofilm, Alexa Fluor 647 dextran dye was added at a final concentration of 1 mM during biofilm culture to label polysaccharides in the extracellular matrix as red, and SYTO-9 fluorescent nucleic acid staining agent at a final concentration of 2.5 mM to label bacterial cells in the biofilm as green. 3D biofilm imaging was performed using confocal laser scanning microscopy.

[0023] from Figure 20 It can be clearly seen that in the blank group, S.mutans , S.sanguinis and S.oralis It is very dense, with spherical protrusions, and contains a rich variety of bacteria and EPS, which can be observed in the Cur@Fe,Mn / CDs@GOx+Glu group. S.mutans , S.sanguinis and S.oralis The biofilm structure remained intact and was not significantly damaged. In the Cur@Fe,Mn / CDs@GOx+Glu+NIR group, the following observations were made. S.mutans and S.sanguinis The biofilm was significantly damaged, with a marked decrease in both bacterial count and residual extracellular matrix. The damage was even more pronounced in *Streptococcus mutans*, but... S.oralis The biofilm structure was not significantly damaged, which further proves that Cur@Fe,Mn / CDs@GOx+Glu can be selectively destroyed under NIR irradiation. S.mutans and S.sanguinis The biofilm structure of pathogenic bacteria reduces the number of bacteria and the amount of extracellular matrix residue.

[0024] The above results indicate that the cascaded nanozyme Cur@Fe,Mn / CDs@GOx prepared in this invention exhibits activity and infrared photothermal properties, and has targeted antibacterial and anti-biofilm effects against cariogenic bacteria.

Claims

1. Application of a curcumin-based carbon dot nanozyme cascade catalytic system in the preparation of near-infrared light-enhanced targeted anti-cariogenic bacteria preparations; The curcumin-based carbon dot nanozyme cascade catalytic system is prepared by mixing curcumin-based carbon dot nanozyme Cur@Fe,Mn / CDs and glucose oxidase GOx in equal mass, sonicating for 60-90 min, and then incubating at 4℃ for 8-10 h. The curcumin-based carbon dot nanozyme Cur@Fe,Mn / CDs is prepared by dissolving 0.5-0.8 g of curcumin in 1-2 mL of anhydrous ethanol, then adding 0.3-0.5 g of citric acid, 0.35-0.50 g of FeCl3·6H2O, and 0.35-0.50 g of... The mixture of MnCl2·4H2O and 30-50 mL of acetic acid aqueous solution with a weight-volume ratio of 1-2% (g:mL) was ultrasonically treated for 20-30 min, and then reacted at 170-190℃ with a microwave power of 1.0-1.2kW for 100-150 min. After cooling the reaction product, it was centrifuged, and the supernatant was filtered through a 0.22 mm filter membrane and vacuum dried to obtain the final product. A curcumin-based carbon dot nanozyme cascade catalytic system is uniformly mixed with glucose. During use, irradiation under 808nm near-infrared light for 10-15 minutes achieves targeted anti-cariogenic bacteria, while inhibiting normal oral streptococci (…). S.oralis No impact; The cariogenic bacteria is Streptococcus mutans ( Streptococcus mutans ) and Streptococcus sanguinis ( Streptococcus sanguinis ).

2. The application according to claim 1, characterized in that: The power of near-infrared light at 808nm is 0.50-1.0W / cm². 2 .

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