Multienzyme active carbon nanodot derived from porphyromonas gingivalis as well as preparation method and application of multienzyme active carbon nanodot
By developing a method for preparing multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis, the problems of multi-enzyme activity and biosafety of nanomaterials in the biomedical field have been solved, enabling the biosafe application of nanomaterials with POD, CAT, and SOD-like catalytic activities.
Patent Information
- Application Number
- CN202511330546.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-17
- Publication Date
- 2025-12-23
AI Technical Summary
Existing nanomaterials have shortcomings in terms of multi-enzyme activity and biosafety in the biomedical field, especially the biosafety of chemically synthesized nanomaterials in vivo needs to be investigated.
Using *Porphyromonas gingivalis* as a biological template, multi-enzyme active carbon nanodots were prepared by hydrothermal reaction and overnight dialysis. Heme was used to provide the active center for the multi-enzyme, and *Porphyromonas gingivalis*-derived multi-enzyme active carbon nanodots were obtained by freeze drying.
The prepared carbon nanodots have a particle size of 4–5 nm and are mainly composed of elements such as carbon, nitrogen, oxygen, phosphorus, sulfur, and iron. They exhibit catalytic activities similar to POD, CAT, and SOD, and demonstrate excellent biosafety.
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Figure CN121180977A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of nanotechnology, specifically to a multi-enzyme active carbon nanodot derived from Porphyromonas gingivalis, its preparation method, and its application. Background Technology
[0002] Against the backdrop of the deep integration of nanotechnology with life sciences, medicine, and other fields, nanomaterials with unique properties are constantly emerging. Among them, carbon nanodots, with their excellent characteristics, have shown great application potential in many cutting-edge fields. As zero-dimensional nanomaterials with a size of less than 10 nm, carbon nanodots have significant advantages such as small particle size, ease of preparation, and low cost. Their abundant oxygen-containing functional groups, such as carbonyl, carboxyl, and hydroxyl groups, endow carbon nanodots with good water solubility and ease of functionalization, making them promising for applications in sensing, bioimaging, light-emitting diodes, and disease treatment. In particular, nanomaterials with enzyme-like activity have broad application prospects in the biomedical field. However, the synthesis of most nanomaterials currently typically uses chemical precursors. As foreign substances, their biocompatibility in vivo needs further investigation. Therefore, there is an urgent need to develop biomaterials with multi-enzyme activity and excellent biocompatibility. Summary of the Invention
[0003] To address the problems existing in the prior art, the present invention aims to provide a multi-enzyme active carbon nanodot derived from Porphyromonas gingivalis and its preparation method. The prepared carbon nanodots exhibit multi-enzyme activity and excellent biocompatibility.
[0004] The present invention also aims to provide the application of multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis in the preparation of products with multi-enzyme catalytic activity.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] This invention provides a method for preparing multi-enzyme active carbon nanodots derived from *Porphyromonas gingivalis*, comprising the following steps: enriching and culturing *Porphyromonas gingivalis* in BHI medium containing heme to prepare a bacterial suspension of *Porphyromonas gingivalis*; subjecting the bacterial suspension of *Porphyromonas gingivalis* to a hydrothermal reaction, centrifuging after the reaction, dialyzing the supernatant overnight, and freeze-drying to obtain multi-enzyme active carbon nanodots derived from *Porphyromonas gingivalis*; the hydrothermal reaction temperature is 120–200℃, and the time is 6–24 h; the dialysis cutoff is 800–5000 kDa.
[0007] Preferably, the BHI culture medium contains 25 μg / mL of heme.
[0008] Preferably, the concentration of the bacterial suspension is 2 × 10⁻⁶. 8CFU / mL ~5×10 8 CFU / mL.
[0009] Preferably, the method for preparing the bacterial suspension includes the following steps: enriching and culturing *Porphyromonas gingivalis* to the logarithmic phase using BHI medium containing heme, collecting the bacterial cells by centrifugation, dispersing them in water, and obtaining the bacterial suspension.
[0010] Preferably, the *Porphyromonas gingivalis* includes any one or more of strains BNCC 353909, BNCC 236547, BNCC 337441, and BNCC 336950.
[0011] Preferably, the centrifugation speed is 10,000 rpm and the time is 10 min.
[0012] Preferably, the freeze-drying temperature is -40 to -80°C and the time is 24 to 48 hours.
[0013] The present invention also provides multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis prepared by the above preparation method.
[0014] The present invention also provides the application of the above-mentioned Porphyromonas gingivalis-derived multi-enzyme active carbon nanodots in the preparation of products with multi-enzyme catalytic activity.
[0015] Preferably, the multi-enzyme catalytic activity includes POD-like catalytic activity, CAT-like catalytic activity, and SOD-like catalytic activity.
[0016] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:
[0017] The multi-enzyme active carbon nanodots derived from *Porphyromonas gingivalis* prepared in this invention are carbon nanodots with a small amount of iron doping and a particle size of approximately 4–5 nm. They are mainly composed of elements such as carbon, nitrogen, oxygen, phosphorus, sulfur, and iron, and have a crystal structure similar to graphene. They possess multi-enzyme activity, including peroxidase-like (POD), catalase-like (CAT), and superoxide dismutase-like (SOD) activities, and exhibit excellent biocompatibility. Attached Figure Description
[0018] Figure 1 TEM image of multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis;
[0019] Figure 2 Particle size distribution of multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis;
[0020] Figure 3 XPS image of multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis;
[0021] Figure 4 XRD pattern of multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis;
[0022] Figure 5 : POD-like catalytic activity diagram of multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis;
[0023] Figure 6 Comparison of POD-like catalytic activities of multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis;
[0024] Figure 7 : CAT-like catalytic activity diagram of multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis;
[0025] Figure 8 SOD-like catalytic activity diagram of multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis;
[0026] Figure 9 : Cellular MTT assay results of multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis;
[0027] Figure 10 : Hemolysis results of multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis. Detailed Implementation
[0028] This invention provides a method for preparing multi-enzyme active carbon nanodots derived from *Porphyromonas gingivalis*, comprising the following steps: enriching and culturing *Porphyromonas gingivalis* in BHI medium containing heme to prepare a bacterial suspension of *Porphyromonas gingivalis*; subjecting the bacterial suspension of *Porphyromonas gingivalis* to a hydrothermal reaction, centrifuging after the reaction, dialyzing the supernatant overnight, and freeze-drying to obtain multi-enzyme active carbon nanodots derived from *Porphyromonas gingivalis*; the hydrothermal reaction temperature is 120–200℃, and the time is 6–24 h; the dialysis cutoff is 800–5000 kDa.
[0029] The preparation method of the bacterial suspension of the present invention includes the following steps: *Porphyromonas gingivalis* is enriched and cultured to the logarithmic growth phase using BHI medium containing heme; the bacterial cells are collected by centrifugation and dispersed in water to obtain a bacterial suspension. The preferred heme concentration in the BHI medium of the present invention is 25 μg / mL. The enrichment culture of the present invention is preferably anaerobic culture at 37℃, and the preferred culture time is 36 h. The preferred centrifugation speed of the present invention is 10000 rpm, and the preferred centrifugation time is 10 min. The concentration of the bacterial suspension of the present invention is 2 × 10⁻⁶. 8 CFU / mL ~5×10 8 CFU / mL, preferably 3×10⁻⁶ 8 CFU / mL or 4×10 8 CFU / mL.
[0030] The *Porphyromonas gingivalis* strain described in this invention includes any one or more of the following strains: BNCC 353909, BNCC 236547, BNCC337441, and BNCC 336950.
[0031] This invention involves a hydrothermal reaction of a bacterial suspension of *Porphyromonas gingivalis*. The preferred temperatures for the hydrothermal reaction are 120°C, 160°C, and 200°C, and the preferred reaction times are 6 hours, 12 hours, and 24 hours. The apparatus for the hydrothermal reaction is preferably a stainless steel hydrothermal reactor lined with polytetrafluoroethylene (PTFE).
[0032] In this invention, after the reaction is completed, centrifugation is performed to obtain the supernatant. The preferred centrifugation speed is 10,000 rpm; the preferred centrifugation time is 10 min.
[0033] The preferred dialysis cutoff values described in this invention are 1500 kDa, 2000 kDa, 3000 kDa, or 4000 kDa.
[0034] The freeze-drying method described in this invention is preferably vacuum freeze-drying, wherein the vacuum degree is preferably 1 Pa, the temperature is preferably -40 to -80°C, and the time is preferably 24 to 48 hours.
[0035] This invention also provides *Porphyromonas gingivalis*-derived multi-enzyme active carbon nanodots prepared by the above-described method. In this invention, *Porphyromonas gingivalis* is cultured in BHI medium containing heme. The Fe-N4 active site of heme provides multi-enzyme activity for the prepared carbon nanodots. In this invention, *Porphyromonas gingivalis* takes up exogenous heme and uses the ingested endogenous heme as an iron source. The active site of heme is retained during the synthesis process, providing a basis for the multi-enzyme activity of the nanodots. Furthermore, *Porphyromonas gingivalis*, used in this invention, is a low-abundance opportunistic pathogen in the oral cavity, widely present in approximately 25% of healthy individuals without oral diseases, ensuring the good biosafety of the synthesized nanodots.
[0036] The present invention also provides the application of the above-mentioned Porphyromonas gingivalis-derived multi-enzyme active carbon nanodots in the preparation of products with multi-enzyme catalytic activity, wherein the multi-enzyme catalytic activity includes POD-like catalytic activity, CAT-like catalytic activity and SOD-like catalytic activity.
[0037] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.
[0038] Unless otherwise specified, the following embodiments are all conventional methods.
[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0040] Example 1
[0041] A method for preparing multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis:
[0042] Porphyromonas gingivalis (BNCC 353909) was enriched and cultured under anaerobic conditions at 37℃ using BHI medium containing 25 μg / mL heme as the nutrient. After culturing for 36 h, once the bacteria had reached the logarithmic growth phase, the well-grown bacteria were collected by centrifugation (10000 rpm, 10 min) and dispersed in water to obtain 2 × 10⁻⁶ bacteria. 8 A bacterial suspension of CFU / mL was prepared. The bacterial suspension was transferred to a stainless steel hydrothermal reactor lined with PTFE and heated at 200°C for 6 h. After the reaction, the supernatant was collected by centrifugation (10000 rpm, 10 min), dialyzed overnight (12 h) using a dialysis bag with an 800 kDa cutoff, and then freeze-dried (-40°C, 48 h) using a vacuum freeze dryer to obtain multi-enzyme active carbon nanodots (P-dots) derived from *Porphyromonas gingivalis*.
[0043] Example 2
[0044] A method for preparing multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis:
[0045] Porphyromonas gingivalis (BNCC 353909) was enriched and cultured under anaerobic conditions at 37℃ using BHI medium containing 25 μg / mL heme as the nutrient. After culturing for 36 h, once the bacteria had reached the logarithmic growth phase, the well-grown bacteria were collected by centrifugation (10000 rpm, 10 min) and dispersed in water to obtain 5 × 10⁻⁶ cells / mL. 8 A bacterial suspension of CFU / mL was prepared. The bacterial suspension was transferred to a stainless steel hydrothermal reactor lined with PTFE and heated at 120°C for 24 h. After the reaction, the supernatant was collected by centrifugation (10000 rpm, 10 min) and dialyzed overnight (12 h) using a dialysis bag with a 5000 kDa cutoff. The supernatant was then freeze-dried (-80°C, 24 h) using a vacuum freeze dryer to obtain multi-enzyme active carbon nanodots (P-dots) derived from *Porphyromonas gingivalis*.
[0046] Example 3
[0047] A method for preparing multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis:
[0048] Porphyromonas gingivalis (BNCC 353909) was enriched and cultured under anaerobic conditions at 37℃ using BHI medium containing 25 μg / mL heme as the nutrient. After culturing for 36 h, once the bacteria had reached the logarithmic growth phase, the well-grown bacteria were collected by centrifugation (10000 rpm, 10 min) and dispersed in water to obtain 2 × 10⁻⁶ bacteria. 8 A bacterial suspension of CFU / mL was prepared. The bacterial suspension was transferred to a stainless steel hydrothermal reactor lined with PTFE and heated at 200°C for 12 h. After the reaction, the supernatant was collected by centrifugation (10000 rpm, 10 min) and dialyzed overnight (12 h) using a dialysis bag with a 1500 kDa cutoff. The supernatant was then freeze-dried (-80°C, 36 h) using a vacuum freeze dryer to obtain multi-enzyme active carbon nanodots (P-dots) derived from *Porphyromonas gingivalis*.
[0049] Example 4
[0050] A method for preparing multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis:
[0051] Porphyromonas gingivalis (BNCC 236547) was enriched and cultured under anaerobic conditions at 37℃ using BHI medium containing 25 μg / mL heme as the nutrient. After culturing for 36 h, when the bacteria reached the logarithmic growth phase, the well-grown bacteria were collected by centrifugation (10000 rpm, 10 min) and dispersed in water to obtain 2 × 10⁻⁶ bacteria. 8 A bacterial suspension of CFU / mL was prepared. The bacterial suspension was transferred to a stainless steel hydrothermal reactor lined with PTFE and heated at 160°C for 12 h. After the reaction, the supernatant was collected by centrifugation (10000 rpm, 10 min) and dialyzed overnight (12 h) using a dialysis bag with a 2000 kDa cutoff. The supernatant was then freeze-dried (-80°C, 36 h) using a vacuum freeze dryer to obtain multi-enzyme active carbon nanodots (P-dots) derived from *Porphyromonas gingivalis*.
[0052] Example 5
[0053] A method for preparing multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis:
[0054] Porphyromonas gingivalis (BNCC 337441) was enriched and cultured under anaerobic conditions at 37℃ using BHI medium containing 25 μg / mL heme as the nutrient. After 36 h of culture, once the bacteria had reached the logarithmic growth phase, the well-grown bacteria were collected by centrifugation (10000 rpm, 10 min) and dispersed in water to obtain 5 × 10⁻⁶ cells / mL. 8A bacterial suspension of CFU / mL was prepared. The bacterial suspension was transferred to a stainless steel hydrothermal reactor lined with PTFE and heated at 200°C for 12 h. After the reaction, the supernatant was collected by centrifugation (10000 rpm, 10 min) and dialyzed overnight (12 h) using a dialysis bag with a 2000 kDa cutoff. The supernatant was then freeze-dried (-80°C, 36 h) using a vacuum freeze dryer to obtain multi-enzyme active carbon nanodots (P-dots) derived from *Porphyromonas gingivalis*.
[0055] Example 6
[0056] A method for preparing multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis:
[0057] Porphyromonas gingivalis (BNCC 336950) was enriched and cultured under anaerobic conditions at 37℃ using BHI medium containing 25 μg / mL heme as the nutrient. After culturing for 36 h, once the bacteria had reached the logarithmic growth phase, the well-grown bacteria were collected by centrifugation (10000 rpm, 10 min) and dispersed in water to obtain 2 × 10⁻⁶ bacteria. 8 A bacterial suspension of CFU / mL was prepared. The bacterial suspension was transferred to a stainless steel hydrothermal reactor lined with PTFE and heated at 200°C for 12 h. After the reaction, the supernatant was collected by centrifugation (10000 rpm, 10 min) and dialyzed overnight (12 h) using a dialysis bag with a 2000 kDa cutoff. The supernatant was then freeze-dried (-80°C, 36 h) using a vacuum freeze dryer to obtain multi-enzyme active carbon nanodots (P-dots) derived from *Porphyromonas gingivalis*.
[0058] Test case
[0059] The multi-enzyme active carbon nanodots (P-dots) derived from *Porphyromonas gingivalis* prepared in Example 3 were validated:
[0060] 1. The prepared P-dots were observed using a transmission electron microscope (TEM) on a Talos F200S. TEM images are shown below. Figure 1 As shown in the figure, the scale bar is 100 nm. The results show that the microstructure of P-dots is a uniformly distributed nanoparticle.
[0061] 2. TEM images of the prepared P-dots were taken using Nano Measurer software. Figure 1 The analysis yielded the following particle size distribution results: Figure 2 As shown in the figure. The results show that the particle size of the prepared P-dots ranges from 3 to 6.5 nm, mainly distributed in the range of 4 to 5.5 nm, and the particle size conforms to a normal distribution.
[0062] 3. The prepared P-dots were analyzed using an ESCALAB 250Xi X-ray photoelectron spectroscopy system. The XPS results are as follows: Figure 3 As shown in the figure. The results show that the prepared P-dots are mainly composed of elements such as C, N, O, P, S, and Fe. The fine spectrum of Fe further confirms the successful incorporation of Fe.
[0063] 4. The prepared P-dots were analyzed using a Bruker-D8 ADVANCE X-ray diffractometer. The XRD results are as follows: Figure 4 As shown in the figure. The results show that the prepared P-dots exhibit broad diffraction at 21.3°, indicating that the material has a high degree of graphitization.
[0064] 5. Verification of multi-enzyme activity:
[0065] (1) POD-like catalytic activity:
[0066] Peroxidase catalyzes the production of reactive oxygen species (ROS) from hydrogen peroxide (H₂O₂). These ROS oxidize colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue ox-TMB, with a maximum absorption at 652 nm. This experiment used a TMB-assisted colorimetric reaction to evaluate the POD-like catalytic activity of P-dots: an acidic phosphate buffer (20 mM, pH = 4) containing 1 mM TMB was prepared as control group 1 (TMB). Then, H₂O₂ (20 mM) and P-dots (100 μg / mL) were added to the solution system of control group 1 to form the H₂O₂ group (control group 2, TMB + H₂O₂) and the P-dots group (control group 3, TMB + P-dots), respectively. Similarly, H₂O₂ (20 mM) and P-dots (100 μg / mL) were added to the solution system of control group 1 to form the experimental group (TMB + H₂O₂ + P-dots). After thorough mixing, each group was incubated at room temperature for 1 hour. The color change of the mixture was observed visually, and its ultraviolet-visible absorption spectrum (500–800 nm) was recorded. The results are as follows: Figure 5 As shown.
[0067] P-dots synthesized under different reaction conditions (120℃, 12h; 160℃, 12h; 200℃, 12h; 200℃, 6h; 200℃, 24h; all other conditions were the same as in Example 3) were prepared into solutions of the same concentration (100 μg / mL). The P-dots catalytic activity was compared according to the experimental steps described above. After co-incubation at room temperature for 1h, the absorbance at 652 nm was recorded. The results are as follows: Figure 6 As shown.
[0068] (2) CAT-like catalytic activity:
[0069] Catalase (CAT) catalyzes the decomposition of H₂O₂ into oxygen and water. This experiment directly evaluated the CAT-like catalytic activity of P-dots using a dissolved oxygen analyzer: Mixtures containing 100 mM H₂O₂ and different concentration gradients of P-dots were prepared (solvent: deionized water, concentrations of 0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 250 μg / mL). After mixing the reaction systems and allowing them to react at room temperature for 30 min, the oxygen content in the different mixtures was directly measured using a dissolved oxygen analyzer. The change in oxygen content in the mixtures reflected the CAT-like catalytic activity of P-dots. Results are as follows: Figure 7 As shown.
[0070] (3) SOD-like catalytic activity:
[0071] In the presence of oxidizing substances, riboflavin can produce superoxide anions (O2) under light irradiation. - O2 - Nitroblue tetrazolium (NBT) can be reduced to blue formazan, which has maximum absorption at 560 nm. Superoxide dismutase (SOD) can scavenge O2. - This inhibits the formation of blue formazan. The SOD-like catalytic activity of P-dots was evaluated by calculating the inhibitory effect of NBT photoreduction: Mixtures containing methionine (36 mM), riboflavin (20 μM), NBT (75 μM), and different concentration gradients of P-dots were prepared beforehand (solvent: deionized water, concentrations of 0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 250 μg / mL, respectively). After thorough mixing, the mixtures were irradiated under white light for 15 min, and the color changes of the different mixtures were observed visually, with their UV-Vis absorption spectra (400–900 nm) recorded. The results are as follows: Figure 8 As shown.
[0072] The results showed that the prepared Porphyromonas gingivalis-derived multienzyme active carbon nanodots (P-dots) had peroxidase-like (POD) activity, catalase-like (CAT) activity, and superoxide dismutase-like (SOD) activity.
[0073] 6. In vitro toxicity verification:
[0074] (1) Cellular MTT assay:
[0075] Mouse fibroblasts (L929 cells) were seeded in 96-well plates and incubated overnight at a density of 10 cells per well. 4Cells were cultured at a concentration of 100 μg / mL. After cell adhesion, P-dots at varying concentrations (final concentrations of 0 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL) were added to the culture medium and co-cultured for 12 h. Subsequently, 10 μL of LTT solution (5 mg / mL) was added to each well and incubated overnight. Afterward, the culture medium was aspirated from all wells, and the formazan was dissolved in DMSO (200 μL per well). The absorbance of all wells at 490 nm was analyzed using a microplate reader. The results are shown below. Figure 9 As shown in the figure. The results showed that when the concentration of P-dots was as high as 100 μg / mL, the cell viability remained good and there was no significant difference compared with the control group.
[0076] (2) Hemolysis test:
[0077] Fresh blood was collected from SD rats and stabilized with heparin. Red blood cells were obtained by centrifugation for further use. The diluted red blood cell suspension (1×10⁻⁶) was then prepared. 8 Red blood cells (p-dots / mL) were mixed with equal volumes of water, physiological saline, and P-dots solutions containing concentration gradients (physiological saline, concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively) to establish positive groups (hemolysis group, i.e., red blood cell aqueous suspension), negative groups (red blood cell physiological saline suspension), and experimental groups (red blood cell P-dots physiological saline suspension). All samples were incubated together at room temperature for 3 hours, centrifuged, and the absorbance of the supernatant was measured at 541 nm.
[0078] Hemolysis rate (%) = [(ODsample - ODnegative) / (ODpositive - ODnegative)] × 100, where ODpositive, ODnegative, and ODsample are the absorbance at 541 nm for the positive, negative, and experimental groups, respectively. Hemolysis results are as follows: Figure 10 As shown, the results indicate that no obvious hemolysis occurred even when the concentration of P-dots was as high as 100 μg / mL.
[0079] The results showed that the prepared Porphyromonas gingivalis-derived multienzyme active carbon nanodots (P-dots) had good biosafety.
[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing multi-enzyme active carbon nanodots derived from *Porphyromonas gingivalis*, characterized in that, Includes the following steps: Porphyromonas gingivalis was enriched and cultured in BHI medium containing heme to prepare a bacterial suspension of Porphyromonas gingivalis. The bacterial suspension of Porphyromonas gingivalis was subjected to a hydrothermal reaction. After the reaction was completed, the suspension was centrifuged, the supernatant was dialyzed overnight, and then freeze-dried to obtain multi-enzyme active carbon nanodots derived from Porphyromonas gingivalis. The hydrothermal reaction is carried out at a temperature of 120–200°C for a duration of 6–24 hours. The dialysis cutoff is 800–5000 kDa.
2. The preparation method according to claim 1, characterized in that, The BHI culture medium contains 25 μg / mL of heme.
3. The preparation method according to claim 1, characterized in that, The concentration of the bacterial suspension is 2×10⁻⁶. 8 CFU / mL ~5×10 8 CFU / mL.
4. The preparation method according to claim 1, characterized in that, The method for preparing the bacterial suspension includes the following steps: enriching and culturing *Porphyromonas gingivalis* to the logarithmic phase using BHI medium containing heme, collecting the bacterial cells by centrifugation, dispersing them in water, and obtaining the bacterial suspension.
5. The preparation method according to claim 1, characterized in that, The *Porphyromonas gingivalis* includes any one or more of strains BNCC353909, BNCC 236547, BNCC 337441, and BNCC 336950.
6. The preparation method according to claim 1, characterized in that, The centrifugation speed was 10,000 rpm and the time was 10 min.
7. The preparation method according to claim 1, characterized in that, The freeze-drying temperature is -40 to -80°C, and the time is 24 to 48 hours.
8. The multi-enzyme activated carbon nanodots derived from Porphyromonas gingivalis prepared by the preparation method according to any one of claims 1 to 7.
9. The application of the multi-enzyme active carbon nanodots derived from *Porphyromonas gingivalis* as described in claim 8 in the preparation of products with multi-enzyme catalytic activity.
10. The application according to claim 9, characterized in that, The multi-enzyme catalytic activity includes POD-like catalytic activity, CAT-like catalytic activity, and SOD-like catalytic activity.