Preparation method and application of near-infrared light enhanced response Cu, Fe-Lyz / AgNPs nano-enzyme

Abstract

The invention discloses a preparation method of a near-infrared light enhanced response Cu, Fe-Lyz / AgNPs nano-enzyme, according to the method, Cu and Fe doped carbon dots are used as a reducing agent to prepare silver nanoparticles, then the Cu, Fe-Lyz / AgNPs nano-enzyme with positive charges on the surface is prepared through modification of lysozyme, and the Cu, Fe-Lyz / AgNPs nano-enzyme has dual activities similar to peroxidase and glutathione oxidase and photo-thermal performance, and can be used for preparing the near-infrared light enhanced response Cu, Fe-Lyz / AgNPs nano-enzyme. Meanwhile, the POD-like activity of the nano-enzyme can be obviously enhanced by the near-infrared light; cu and Fe-Lyz / AgNPs have a relatively good antibacterial effect on escherichia coli and staphylococcus aureus; meanwhile, based on the enhancement effect of the penicillin G potassium on the activity of Cu and Fe-Lyz / AgNPs oxidase, the invention establishes a rapid colorimetric detection method for the penicillin G potassium, when the method is used for detecting the penicillin G potassium in plasma and milk powder samples, the detection limit is as low as 0.05 mu g / mL, and the method has relatively high reliability and accuracy.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a preparation method of a near-infrared light-enhanced response Cu, Fe-Lyz / AgNPs nanoscale enzyme and application of the Cu, Fe-Lyz / AgNPs nanoscale enzyme in detection of penicillin G potassium and preparation of an antibacterial agent. BACKGROUND

[0002] The increasing incidence of bacterial infections has become a global concern, threatening human life and health. More worrying is that the misuse of antibiotics has led to an increase in bacterial drug resistance, and traditional antibiotics have become increasingly weak in their effects on bacteria, so it is crucial to develop new and effective antibacterial agents. Among various antibacterial materials, carbon dots (CDs) have received extensive attention as a new alternative to traditional antibacterial agents due to their simple synthesis, microbial attachment, low toxicity, and good biocompatibility. In addition, CDs have the ability to mimic enzyme activity and produce toxic reactive oxygen species (ROS), and metal doping can improve ROS production to achieve effective antibacterial effects. At the same time, CDs have excellent optical properties, especially metal-doped CDs respond to near-infrared light (NIR), producing a unique photothermal / photodynamic synergistic effect, providing a broader application space for the antibacterial application of this type of material. The material can effectively utilize the deep tissue penetration ability of NIR light to achieve precise and controllable sterilization effects, while significantly reducing damage to normal tissues. In addition, NIR-responsive materials have shown significant advantages in anti-infection therapy, such as high-efficiency sterilization and reduced drug resistance, bringing a revolutionary new approach to the field of anti-infection therapy.

[0003] Penicillin G potassium (PGK) is a broad-spectrum beta-lactam antibiotic widely used to treat bacterial infections in humans and animals. However, there is a strong awareness that the overuse of antibiotics poses a significant risk to the ecosystem and human health, as unmetabolized antibiotics can enter water or soil, causing environmental pollution, and there is growing concern about antibiotic resistance. On February 27, 2017, the World Health Organization (WHO) released a list of the "12 most dangerous drug-resistant bacteria", and bacteria resistant to beta-lactam antibiotics were listed as the most dangerous. Since PGK is one of the most common beta-lactam drugs detected in the environment, there is a need for effective methods to achieve sensitive detection of PGK in environmental samples. SUMMARY

[0004] The application provides a preparation method of near-infrared light enhanced response Cu, Fe-Lyz / AgNPs nanoscale enzyme and application of the Cu, Fe-Lyz / AgNPs nanoscale enzyme in detection of penicillin G potassium and preparation of antibacterial agents. The Cu, Fe-Lyz / AgNPs nanoscale enzyme with positive charges on the surface is prepared by using Cu, Fe doped carbon dots as a reducing agent to prepare silver nanoparticles and then modifying the silver nanoparticles by lysozyme. The Cu, Fe-Lyz / AgNPs nanoscale enzyme has dual activities of peroxidase-like (POD-like) and glutathione oxidase-like (GSH oxidase-like) and photo-thermal performance. Meanwhile, near-infrared light (NIR) significantly enhances the POD-like activity of the nanoscale enzyme. Research results show that the Cu, Fe-Lyz / AgNPs nanoscale enzyme has good antibacterial effect on escherichia coli and staphylococcus aureus. After 7 minutes of treatment of the Cu, Fe-Lyz / AgNPs nanoscale enzyme and NIR irradiation, nearly 100% of pathogenic bacteria can be inactivated and more than 90% of biofilms can be inhibited, and the minimum inhibitory concentrations are 5 mu g / mL and 10 mu g / mL respectively. These effects are attributed to the generation of active oxygen of the Cu, Fe-Lyz / AgNPs nanoscale enzyme, the release of silver ions, the depletion of glutathione (GSH) and the positive charges on the surface. Meanwhile, based on the fact that penicillin G potassium can enhance the oxidase-like activity of the Cu, Fe-Lyz / AgNPs nanoscale enzyme, rapid colorimetric detection of penicillin G potassium is realized. When the method is used for detection of penicillin G potassium in plasma and milk powder samples, the detection limit is as low as 0.05 mu g / mL, and the method has high reliability and accuracy.

[0005] The preparation method of the near-infrared light enhanced response Cu, Fe-Lyz / AgNPs nanoscale enzyme is as follows: (1) 0.100-0.150g tartaric acid, 0.100-0.150g L-tryptophan, 0.200-0.300g CuCl2.2H2O and 0.200-0.300g FeCl3.6H2O are added into 20-30mL deionized water, ultrasonic treatment is carried out for 20-30min, the mixture is transferred to a Teflon autoclave, and the autoclave is placed in a muffle furnace, heated at 120-140 DEG C for 10-12h, cooled to room temperature, centrifuged, the supernatant is filtered through a 0.22mm filter membrane, and vacuum drying is carried out to obtain Cu, Fe doped carbon dots Cu, Fe-CDs; The centrifugation is carried out at 8000-10000r / min for 5-10min; (2) 0.2 mg / mL iron copper doped carbon dots Cu,Fe-CDs solution 350-500 μL, 2.2 mg / mL AgNO3 solution 1.0-1.5 mL, 50 mg / mL lysozyme 100-200 μL are added into 20-30 mL deionized water, stirred at room temperature for 20-30 min, vacuum dried, and Cu,Fe-Lyz / AgNPs nanoscale enzyme is prepared.

[0006] The Cu,Fe-Lyz / AgNPs nanoscale enzyme prepared by the above method is applied in the detection of penicillin G potassium, specifically, Cu,Fe-Lyz / AgNPs nanoscale enzyme solution, 3,3',5,5'-tetramethylbenzidine (TMB) solution and H2O2 solution are sequentially added into penicillin G potassium solution with different concentrations, and then the volume is adjusted with pH 4.0 acetate buffer solution, and the reaction is carried out at room temperature for 10-20 min, and then the absorbance value is measured at 654 nm wavelength to determine the linear relationship between the concentration of penicillin G potassium and the absorbance value, and the regression equation is obtained. The Cu,Fe-Lyz / AgNPs nanoscale enzyme solution, 3,3',5,5'-tetramethylbenzidine solution and H2O2 solution are added into the sample to be tested, and then the volume is adjusted with pH 4.0 acetate buffer solution, and the reaction is carried out at room temperature for 10-20 min, and then the absorbance value is measured at 654 nm wavelength, and the regression equation is introduced to obtain the penicillin G potassium content of the sample to be tested.

[0007] The concentration of the Cu,Fe-Lyz / AgNPs nanoscale enzyme solution is 1 mg / mL, and the addition amount is 10-20 μL; the concentration of the 3,3',5,5'-tetramethylbenzidine solution is 5 mmol / L, and the addition amount is 20-40 μL; the concentration of the H2O2 solution is 50 mmol / L, and the addition amount is 20-40 μL; and the concentration of the pH 4.0 acetate buffer solution is 0.1 mol / L.

[0008] The Cu,Fe-Lyz / AgNPs nanoscale enzyme prepared by the above method is applied in the preparation of an antibacterial agent, and the Cu,Fe-Lyz / AgNPs nanoscale enzyme realizes antibacterial effect under the action of peroxide and near-infrared light irradiation; the Cu,Fe-Lyz / AgNPs nanoscale enzyme is applied to the removal of E. coli, Staphylococcus aureus and their biofilms.

[0009] The power of the 808 nm near-infrared light is 0.50-1.0 W / cm 2 .

[0010] Advantages and technical effects of the present application: 1. This invention uses Cu,Fe-doped carbon dots as a reducing agent to prepare silver nanoparticles, which are then modified with lysozyme to obtain positively charged Cu,Fe-Lyz / AgNPs nanozymes. These Cu,Fe-Lyz / AgNPs nanozymes possess dual activities of peroxidase-like (POD-like) and glutathione oxidase-like (GSH-like) and photothermal properties. Near-infrared light (NIR) significantly enhances the POD-like activity of the nanozymes. This dual-enzyme synergistic system can synergistically catalyze the conversion of H2O2 and endogenous glutathione (GSH) into highly cytotoxic hydroxyl radicals (·OH) and oxidized glutathione (GSSG). Through a dual-pathway mechanism of oxidative stress, combined with the material's excellent photothermal properties and positive surface charge, it facilitates binding with negatively charged bacteria, achieving control of Gram-negative bacteria (such as...). E. coli Gram-positive bacteria (such as...) S. aureus It has excellent effects in killing and removing biofilms; 2. In vitro experiments showed that Cu,Fe-Lyz / AgNPs nanozymes possess excellent biocompatibility and low toxicity. Combined with their POD-like activity and photothermal properties, these nanozymes exhibit broad-spectrum antibacterial activity under NIR activation, effectively inactivating Gram-negative bacteria. E. coli 5 μg / mL, 99.9%), Gram-positive bacteria ( S. aureus 10 μg / mL, 99.8%); 50 μg / mL Cu,Fe-Lyz / AgNPs nanozymes can make E. coli and S. aureus Biofilms decreased to around 10%; this "capture-catalysis-kill" three-modal synergistic system provides a new model for developing smart nanomaterials that resist microbial resistance. 3. Based on the interaction between potassium penicillin G and Cu,Fe-Lyz / AgNPs nanozyme, which leads to a significant enhancement of enzyme POD-like activity, this invention establishes a new colorimetric detection method for potassium penicillin G. When the established method is applied to the detection of potassium penicillin G in plasma and milk powder samples, the detection limit is as low as 0.05 μg / mL, which has high reliability and accuracy. In summary, the Cu,Fe-Lyz / AgNPs nanozyme of this invention can achieve effective antibacterial activity against Escherichia coli and Staphylococcus aureus, as well as visualized detection of potassium penicillin G. Attached Figure Description

[0011] Figure 1 The images are TEM images of the Cu,Fe-Lyz / AgNPs nanozyme prepared in Example 1 of this invention. Image a is 20 nm and image b is 5 nm. Figure 2The Cu 2p high-resolution XPS spectrum of the Cu,Fe-Lyz / AgNPs nanozyme prepared in Example 1 of this invention; Figure 3 The high-resolution XPS spectrum of Fe 2p of the Cu,Fe-Lyz / AgNPs nanozyme prepared in Example 1 of this invention; Figure 4 for E. coli , Cu,Fe / AgNPs, Cu,Fe-Lyz / AgNPs, Cu,Fe-Lyz / AgNPs+ E. coli The results of the Zeta potential characterization are shown in the figure. Figure 5 The UV-Vis absorption spectra of Cu,Fe-Lyz / AgNPs nanozymes and Cu,Fe-Lyz / AgNPs nanozymes + NIR are shown. Figure 6 Figure a shows the temperature rise curves of Cu,Fe-Lyz / AgNPs nanozymes. Figure a shows the temperature rise curves of Cu,Fe-Lyz / AgNPs nanozymes at different concentrations under the same power (1.0 W / cm²). 2 Figure b shows the temperature rise curves of Cu,Fe-Lyz / AgNPs nanozymes at different powers. Figure 7 The photothermal conversion rate curves of the 150 μg / mL Cu,Fe-Lyz / AgNPs nanozyme solution are shown in Figure a. The heating and cooling curves are shown in Figure b. The linear fitting of ln(θ) - time during the cooling period is shown in Figure b. Figure 8 For the reaction system Cu,Fe-Lyz / AgNPs + H2O2 + TMB at 1.0 W / cm 2 Ultraviolet absorption spectra under 808nm irradiation or no irradiation conditions; Figure 9 The steady-state kinetic analysis results of Cu,Fe-Lyz / AgNPs nanozymes on TMB are shown in Figure a, where no irradiation is observed and Figure b shows the results of infrared light irradiation. Figure 10 The steady-state kinetic analysis results of Cu,Fe-Lyz / AgNPs nanozymes on H2O2 are shown in Figure a, where no irradiation is observed and Figure b shows the results under infrared light irradiation. Figure 11 Figure a shows the results of GSH oxidase-like enzyme activity assay of Cu,Fe-Lyz / AgNPs nanozymes (Figure a) and the effect of different concentrations of Cu,Fe-Lyz / AgNPs nanozymes on GSH oxidase-like enzyme activity (Figure b). Figure 12The influence of different concentrations of Cu, Fe-Lyz / AgNPs nanoszyme on the survival rate of Hep G2 cells (a) and HUVECs cells (b) at 24h and 48h, the MIC in the figure is 10 μg / mL; Figure 13 The antibacterial performance detection results of different experimental groups under 808nm irradiation and no irradiation on different pathogenic bacteria (a), and the influence of different concentrations of Cu, Fe-Lyz / AgNPs nanoszyme on the antibacterial performance of different pathogenic bacteria under 808nm irradiation and no irradiation (b); Figure 14 The antibacterial performance statistical results of different concentrations of Cu, Fe-Lyz / AgNPs nanoszyme on E. coli (a) and S. aureus (b); Figure 15 The CV staining results of different concentrations of Cu, Fe-Lyz / AgNPs nanoszyme on inhibiting E. coli and S. aureus biofilm; Figure 16 The inhibition rate statistical results of different concentrations of Cu, Fe-Lyz / AgNPs nanoszyme on inhibiting E. coli (a) and S. aureus (b) biofilm; Figure 17 The SEM images of Cu, Fe-Lyz / AgNPs nanoszyme treated different bacteria; Figure 18 The UV-visible spectrum of H2O2+Cu, Fe-Lyz / AgNPs, H2O2+PGK+Cu, Fe-Lyz / AgNPs; Figure 19 The hydroxyl radical capture diagram of H2O2+Cu, Fe-Lyz / AgNPs, H2O2+PGK+Cu, Fe-Lyz / AgNPs; Figure 20 The UV-visible absorption spectrum (a) and linear regression equation (b) of PGK; Figure 21 The influence results of coexisting substances on the determination of PGK; Figure 22 The influence results of metal ions on the determination of PGK. DETAILED DESCRIPTION

[0012] The content of the present application is further illustrated by the following examples, but these examples do not limit the protection scope of the present application, and the methods in the examples are all conventional methods, and the reagents used are all conventional commercially available reagents or reagents prepared according to conventional methods, unless otherwise specified; Example 1: Preparation and antibacterial detection of Cu,Fe-Lyz / AgNPs nanozymes 1. Preparation of iron and copper doped carbon dots (Cu,Fe-CDs): 0.100 g tartaric acid, 0.100 g L-tryptophan, 0.200 g CuCl2·2H2O and 0.200 g FeCl3·6H2O were added to 20 mL of deionized water, sonicated for 20 min, transferred to a Teflon autoclave, placed in a muffle furnace, heated at 120 °C for 12 h, cooled to room temperature, centrifuged at 8000 r / min for 10 min, filtered the supernatant through a 0.22 mm filter membrane, and vacuum dried to obtain iron and copper doped carbon dots Cu,Fe-CDs; 2. Preparation of Cu,Fe-Lyz / AgNPs nanozyme: 350 μL of 0.2 mg / mL Cu,Fe-CDs solution, 1.0 mL of 2.2 mg / mL AgNO3 solution, and 100 μL of 50 mg / mL lysozyme were added to 20 mL of deionized water. The mixture was stirred at room temperature for 20 min and then dried under vacuum to obtain Cu,Fe-Lyz / AgNPs nanozyme. At the same time, Cu,Fe / AgNPs nanozymes were prepared as a control, using the same method as above, except that lysozyme was not added. The clustered microsphere morphology of Cu,Fe-Lyz / AgNPs nanozymes was verified by TEM. Figure 1 Image a shows that due to the abundance of oxygen-containing functional groups in CDs, the nanoparticles all exhibit a monodisperse spherical structure without significant aggregation. Meanwhile, high-resolution TEM (HRTEM) images ( Figure 1 b) The material shows clear lattice stripes with interplanar spacings of 0.22 nm, 0.31 nm and 0.25 nm, corresponding to the (111), (211) and (111) lattice spaces of Cu, Fe and Ag, respectively; Figure 2 In the high-resolution spectrum of Cu 2p, Cu was detected at binding energies of 931.7 eV and 952.3 eV. + and Cu 2+ The presence of high-resolution Fe 2p XPS spectra ( Figure 3 It can be decomposed into three peaks, corresponding to Fe, respectively. 2+ (711.6eV), Fe 3+ (717.4 V) and Fe 3+ The satellite peak (724.5 eV); right E. coli Cu,Fe / AgNPs nanozymes, Cu,Fe-Lyz / AgNPs nanozymes, Cu,Fe-Lyz / AgNPs+ E. coli The Zeta potential was characterized, and the results are as follows: Figure 4 , E. coliCu, Fe / AgNPs, Cu, Fe-Lyz / AgNPs and Cu, Fe-Lyz / AgNPs E. coli The Zeta of Cu, Fe / AgNPs, Cu, Fe-Lyz / AgNPs and Cu, Fe-Lyz / AgNPs were -11.3 mV, -9.5 mV, 13.1 mV and 2.6 mV, respectively, which indicated that the Cu, Fe-Lyz / AgNPs nanoscale enzyme prepared by lysozyme modification was positively charged on the surface, and E. coli could be combined on the Cu, Fe-Lyz / AgNPs nanoscale enzyme; 3. Evaluation of the photothermal performance of Cu, Fe-Lyz / AgNPs nanoscale enzyme: In order to evaluate the NIR-triggered photothermal performance of Cu, Fe-Lyz / AgNPs nanoscale enzyme, the temperature change of the nanoscale enzyme solution under 808 nm NIR laser irradiation was measured, and it was found by UV-Vis-NIR absorption spectrum characterization that Cu, Fe-Lyz / AgNPs nanoscale enzyme showed a significant absorption peak at 808 nm wavelength (Fig. 3B). Figure 5 ), which was directly related to its photothermal energy conversion performance.

[0013] The photothermal heating curve confirmed that the temperature rise was related to the concentration of Cu, Fe-Lyz / AgNPs nanoscale enzyme and the infrared intensity (Fig. 3C). Figure 6 Under 808 nm light irradiation, the temperature of Cu, Fe-Lyz / AgNPs nanoscale enzyme solution with a concentration of 200 µg / mL rose to 66.6°C, which could effectively induce bacterial membrane structure damage, and the temperature rise of pure water (Blank) under 808 nm infrared irradiation was negligible, which indicated that Cu, Fe-Lyz / AgNPs nanoscale enzyme had the ability to convert NIR light energy into heat energy; To further provide a basis for the study of the photothermal performance of Cu, Fe-Lyz / AgNPs nanoscale enzyme, the photothermal conversion efficiency (η T ) of Cu, Fe-Lyz / AgNPs nanoscale enzyme solution (150 μg / mL) was calculated according to the following formula:

[0014] In the formula, T max is the equilibrium temperature of the sample solution, T surr corresponds to the ambient temperature of the experiment, Q dis is the heat loss of the container absorbing light, where Q dis = (5.4 × 10 -4 ), I represents the power density of 808 nm laser (1.0 W / cm 2 ), and the absorbance of the sample solution at 808 nm is denoted asA λ To determine the value of hs , the following equation is used:

[0015] where m is the mass of the sample solution, C water corresponding to the heat capacity of water (4.2 J kg -1 K -1 ), τ s is the time constant of the system. Thus, the photothermal conversion efficiency of the Cu,Fe-Lyz / AgNPs nanoszyme solution (150 μg / mL) is 59.6% (η = 0.596), which exhibits a high photothermal conversion rate. Figure 7

[0016] 4. Evaluation of the peroxidase-like activity of Cu,Fe-Lyz / AgNPs nanoszyme: The peroxidase-like (POD-like) activity of Cu,Fe-Lyz / AgNPs nanoszyme was evaluated using TMB as a catalytic reaction substrate. In a 5 mL colorimetric tube, 100 μL of 5 mmol / L TMB, 100 μL of 50 mmol / L H2O2, and 50 μL of 1 mg / mL Cu,Fe-Lyz / AgNPs nanoszyme solution were added, and then 0.1 mol / L HAc-NaAc buffer solution (pH 4.0) was added to a final volume of 3 mL. After mixing thoroughly, the reaction was allowed to proceed for 10 min, after which the absorbance was measured at 654 nm. Meanwhile, the reaction system was irradiated at 1.0 W / cm 2 , 808 nm for 7 min to compare the enzyme activity, and the absorbance was measured at 654 nm. TMB alone, H2O2 alone, and TMB + H2O2 alone were used as controls. The results are shown in Figure 8 When Cu,Fe-Lyz / AgNPs nanoszyme and H2O2 were present in the system, a significant oxidation reaction of TMB occurred, which fully demonstrated that Cu,Fe-Lyz / AgNPs nanoszyme had POD-like catalytic activity and could simulate the function of natural peroxidase to catalyze the oxidation of TMB by H2O2 to generate a blue product. Further, when NIR irradiation was applied to the reaction system for 7 min, it was observed that the POD-like activity of Cu,Fe-Lyz / AgNPs nanoszyme was significantly enhanced by nearly 4-fold, and Cu,Fe-Lyz / AgNPs nanoszyme exhibited the characteristic of near-infrared light-enhanced peroxidase-like activity.

[0017] The Michaelis-Menten catalytic kinetic parameters were also determined (kcat, KM, and kcat / KM). Figure 9 , Figure 10 ​and Table 1), the Michaelis constant of Cu,Fe-Lyz / AgNPs nanoszyme to substrate TMB and H2O2 K m were 1.04 mM and 0.20 mM, respectively, and the reaction rate constant was 3.88 x 10 -8 M / s and 1.20 x 10 -8 M / s, respectively, and the absorbance at 808 nm after infrared light irradiation was K m were 0.23 mM and 0.18 mM, respectively, and the reaction rate constant was 22.4 x 10 -8 M / s and 1.58 x 10 -8 M / s, indicating that infrared light irradiation increased the affinity and reaction rate of Cu,Fe-Lyz / AgNPs nanoszyme to the substrate; Table 1 Michaelis catalytic kinetic parameters ; 5. Evaluation of glutathione oxidase (GSH oxidase-like) activity: 50 μL of 2 mg / mL Cu,Fe-Lyz / AgNPs nanoszyme solution was added to a mixed solution of 100 μL of 100 mM 5,5'-dithiobisnitrobenzoic acid (DTNB) and 1 mL of 2 mg / mL GSH, and the volume was made up to 5 mL with water, and then the change in absorbance at 420 nm was observed, and the results are shown in Figure 11 a. Compared with the blank group, the absorbance value at 420 nm of the reaction system with the addition of nanoszyme decreased significantly, indicating that Cu,Fe-Lyz / AgNPs had significant GSH scavenging activity. In addition, as shown in Figure 11 b, by testing different concentrations of Cu,Fe-Lyz / AgNPs nanoszyme solution under NIR irradiation, it was found that the GSH oxidase-like enzyme activity increased with increasing concentration, indicating that the enzyme activity was positively correlated with the concentration, and the above results all indicated that Cu,Fe-Lyz / AgNPs nanoszyme could effectively oxidize GSH to oxidized glutathione (GSSG).

[0018] 6. Cytotoxicity test: The CCK-8 cell viability kit was used to detect the cytotoxicity of Cu,Fe-Lyz / AgNPs nanoszyme, and human umbilical vein endothelial cells and liver cancer cells (HUVECs and HepG2, Beina Chuanglian Biotechnology Co., Ltd.) were inoculated in a 96-well plate and cultured for 24 h, then incubated with different concentrations of Cu,Fe-Lyz / AgNPs nanoszyme solution for 24 h and 48 h, and then the cells were rinsed with PBS, and CCK-8 solution was added to each well to a concentration of 10%, and incubated at 37°C, and the absorbance was measured at 450 nm; CCK-8 analysis Figure 12 showed that Cu,Fe-Lyz / AgNPs nanoszyme had no toxicity to cells.

[0019] 7. Antibacterial experiment of Cu,Fe-Lyz / AgNP nanozyme The following bacterial strains were obtained from Beina Innovation Biotechnology Co., Ltd. Staphylococcus aureus ( S. aureus ATCC 43300), Escherichia coli ( E. coli ATCC-8099) represents Gram-positive and Gram-negative strains. The antibacterial performance of Cu,Fe-Lyz / AgNPs nanozymes was determined by counting CFUs using the plate count method. First, *Escherichia coli* and *Staphylococcus aureus* were incubated for 24 hours on solid Luria-Bertani (LB) medium and solid nutrient broth medium, respectively. A small number of colonies were picked up with an inoculation loop and inoculated into the corresponding liquid medium (5 mL). After incubation at 37°C and 180 rpm for 12 hours, a bacterial suspension (1×10⁻⁶) was obtained. 8 CFU / mL), diluted to 1×10⁻⁶ with sterile phosphate-buffered saline (PBS). 5 CFU / mL.

[0020] The experimental groups were set as follows: Cu,Fe-Lyz / AgNPs+H2O2 group, Cu,Fe-Lyz / AgNPs+H2O2+NIR group, positive control group (gentamicin or cephalexin), control group with only H2O2 added, and blank control group. The bacterial suspension was added to phosphate buffer as a blank control group. Other experimental groups consisted of bacterial suspension mixed with the corresponding experimental materials. The concentration of Cu,Fe-Lyz / AgNPs was 5 μg / mL, and the concentration of H2O2 was 0.5 mmol / L. After treatment at 1.0 W / cm², the bacterial suspension was... 2 The bacterial suspension was irradiated with 808 nm light for 7 min or without infrared irradiation, and then incubated at 37°C for 30 min. After dilution (100 μL), the suspension was evenly spread on LB solid medium and nutrient broth solid medium, and incubated at 37°C for 24 h. The colony count was calculated to determine the antibacterial performance. The results are as follows: Figure 13 As shown in Figure a, at the same concentration, the Cu,Fe-Lyz / AgNPs+H2O2+NIR group exhibited the best antibacterial effect, and its antibacterial effect against both bacteria was superior to that of the positive control drug. E. coli and S. aureus The positive drugs were gentamicin and cephalexin, respectively. This phenomenon confirms that NIR light irradiation can significantly enhance the antibacterial properties of Cu,Fe-Lyz / AgNPs composite materials. The mechanism may be related to the local thermal effect and ROS increase induced by NIR light.

[0021] Figure 13 b、 Figure 14As shown, in the presence of H2O2, Cu,Fe-Lyz / AgNPs nanoszyme showed significant concentration-dependent antibacterial effect on different microorganisms; for gram-negative bacteria E. coli When the concentration of Cu,Fe-Lyz / AgNPs was 5 μg / mL, the sterilization rate was 99.9%; for gram-positive bacteria S. aureus The inhibition rate reached 99.8% at a concentration of 10 μg / mL.

[0022] To further evaluate the antibiofilm performance of Cu,Fe-Lyz / AgNPs nanoszyme, we explored its antibiofilm ability by crystal violet (CV) staining, and measured the absorbance at 590 nm to quantify the biofilm; different concentrations of Cu,Fe-Lyz / AgNPs nanoszyme, H2O2 (0.5 mmol / L) were mixed with E. coli or S. aureus After 808 nm irradiation for 30 min, they were placed in a constant temperature incubator for 24 h, and the production rate of bacterial biofilm was compared with the blank control group, the group without infrared irradiation, and the control group with only H2O2 added; Figure 15 、 16 The results showed that in the presence of H2O2, increasing the content of Cu,Fe-Lyz / AgNPs nanoszyme led to a decrease in biofilm production, and 100 μg / mL Cu,Fe-Lyz / AgNPs could reduce the biofilm to about 7.13%, E. coli S. aureus 11.75%, indicating that Cu,Fe-Lyz / AgNPs had excellent inhibitory ability on biofilm production; Cu,Fe-Lyz / AgNPs+NIR nanoszyme inhibited the biofilm of E. coli and S. aureus , and the effect of Cu,Fe-Lyz / AgNPs+NIR was better than that of Cu,Fe-Lyz / AgNPs nanoszyme without irradiation.

[0023] Figure 17 Scanning electron microscopy (SEM) characterization revealed that the bacteria in the blank control group showed typical rod-shaped or spherical ultrastructure, maintaining cell membrane integrity, confirming that they were not affected by the nanomaterials. After Cu,Fe-Lyz / AgNPs+H2O2+NIR treatment, the bacteria showed characteristic structural damage: ① membrane surface wrinkles; ② cell collapse; ③ intracellular material leakage. This multi-level structural damage pattern is closely related to the cascade catalytic properties of nanoszyme - the enhanced electron transfer efficiency of NIR increases the quantum yield of H2O2 conversion to ·OH, which in turn triggers a cascade reaction of bacterial membrane lipid peroxidation, revealing the antibacterial action path of the material.

[0024] ​The above results show that the nanoenzyme Cu,Fe-Lyz / AgNPs prepared in the application exhibits high peroxidase activity and photothermal performance under NIR irradiation, and catalyzes H2O2 to generate ROS with bactericidal effect, thereby achieving E. coli and S. aureus good antibacterial effect, and the light response characteristic characterization shows that the nanoenzyme system exhibits a dual-mode antibacterial mechanism under 808nm NIR excitation: ①POD-like activity is improved; ②photothermal conversion efficiency η=59.6%, which can realize local temperature rise. For gram-negative bacteria E. coli , when the concentration of Cu,Fe-Lyz / AgNPs is 5μg / mL, the bactericidal rate is 99.9%; for gram-positive bacteria S. aureus , the inhibition rate reaches 99.8% at a concentration of 10μg / mL.

[0025] 9. Colorimetric detection of penicillin G potassium (PGK) by Cu,Fe-Lyz / AgNPs nanoenzyme (1) Effect of PGK on the POD-like activity of Cu,Fe-Lyz / AgNPs nanoenzyme: In a 96-well plate, add 20μL of 5mmol / L TMB solution, 200μL of 50mmol / L H2O2, 10μL of 1mg / mL Cu,Fe-Lyz / AgNPs nanoenzyme, 50μL of 10μg / mL PGK solution, and 200μL of 0.1mol / L pH 4.0 HAc-NaAc buffer solution, mix well, and react at room temperature for 10min, then measure the absorbance at 654nm, and at the same time, take the blank without PGK as a comparison, and the results are as follows Figure 18 , PGK significantly increases the POD-like activity.

[0026] (2) Detection of hydroxyl radicals (·OH): Terephthalic acid (TA) is used as a probe to detect ·OH, and TA is oxidized to 2-hydroxyterephthalic acid (TAOH) in the presence of ·OH. Add 100μL of 1mg / mL Cu,Fe-Lyz / AgNPs nanoenzyme, 100μL of 5mg / mL TA, and 200μL of 50mmol / L H2O2 to 2.6mL of pH 4.0 HAc-NaAc buffer solution, mix well, and incubate at room temperature for 4h, then measure the fluorescence at 438nm under excitation at 330nm to explore the catalytic activity, and the results are as follows Figure 19 , Cu,Fe-Lyz / AgNPs+H2O2 has strong ability to generate ·OH, and under the same detection conditions, PGK is added for testing, and the results show that the addition of PGK increases the fluorescence intensity of the system, indicating that the addition of PGK is conducive to the generation of ·OH, and it is confirmed that the increase of POD-like activity by PGK is realized by generating more ·OH.

[0027] (3) PGK working curve preparation: add 20 μL of 5 mmol / L TMB solution, 20 μL of 50 mmol / L H2O2, 10 μL of 1 mg / mL Cu,Fe-Lyz / AgNPs nanoscale enzyme, 50 μL of PGK solution with different concentrations, 200 μL of 0.1 mol / L pH 4.0 HAc-NaAc buffer solution in a 96-well plate, mix well, react at room temperature for 10 min, measure the absorbance at 654 nm, set a blank control without adding PGK, and calculate ΔA = A - A0, A0 is the absorbance of the blank control, and A is the absorbance of the PGK added, draw the calibration curve of the absorbance difference ΔA and the PGK concentration, see Figure 20 , the regression equation, the correlation coefficient, the relative standard deviation, and the linear range are shown in Table 2; Table 2 Linear equation, correlation coefficient, relative standard deviation, and linear range .

[0028] (4) Method specificity investigation: replace PGK with other antibiotics (fluconazole (FCN), azithromycin (AZM), amoxicillin (AMX), chloramphenicol (CM), roxithromycin (RXM), ornidazole (ONZ), penicillin sodium (Pen-Na), tinidazole (TNZ), clarithromycin (CLR), sulfamethoxazole (SMZ), and roxidazole (PNZ)), and coexisting ions (Na + , K + , Ca 2+ , Mg 2+ , Cu 2+ , Zn 2+ , Fe 2+ , Cl - , SO4 2- , NO3 - ), detect the influence of PGK and other substances on Cu,Fe-Lyz / AgNPs nanoscale enzyme in the detection system of step (3); wherein the concentration of PGK and other antibiotics is 5 μg / mL, and the concentration of coexisting ions is 50 μg / mL, and the results are shown in Figure 21 、 22 From the figure, it can be seen that the Cu,Fe-Lyz / AgNPs nanoscale enzyme detection system has good selectivity, and PGK significantly enhances the oxidation reaction, and other substances are almost none. The method of the present application has good selectivity for determining PGK.

[0029] (5) PGK determination in samples Milk powder sample: first 2.5g milk powder was dissolved in 20mL water at 25℃, then 8.0mL acetic acid (3%, v / v) was added, stirred vigorously for 15min, diluted with 50mL deionized water, centrifuged at 4000rpm for 15min, and the supernatant was taken as the test solution; Plasma sample: obtained from the hospital of Kunming University of Science and Technology, diluted 50 times with pH 7.0 phosphate buffer solution (PBS, 0.1mol / L) to obtain the test solution; In the above test solution, 0.5, 10, 50μg / mL of PGK standard solution was added, and the recovery rate test of the detection sample was carried out, and the results are shown in Table 3, the results show that the recovery rate of PGK detected in the detection sample is between 93.9%~106.1%, and the RSD is less than 4%, and the results show that the method established by the application can be used for the detection of PGK in milk powder and plasma samples; Table 3 sample recovery rate and RSD (n=3)

[0030] The PGK determination method established by the application has the advantages of less processing steps, short processing time, low processing cost, simple operation, and does not need large instrument equipment, and has strong advantages in actual detection.

Claims

1. A method for preparing near-infrared light-enhanced responsive Cu,Fe-Lyz / AgNPs nanozymes, characterized in that, The steps are as follows: (1) Add 0.100-0.150g tartaric acid, 0.100-0.150g L-tryptophan, 0.200-0.300g CuCl2·2H2O, and 0.200-0.300g FeCl3·6H2O to 20-30mL of deionized water, sonicate for 20-30min, transfer to a Teflon autoclave, place in a muffle furnace, heat at 120-140℃ for 10-12h, cool to room temperature, centrifuge, filter the supernatant through a 0.22mm filter membrane, and vacuum dry to obtain iron-copper doped carbon dots Cu,Fe-CDs; (2) Add 350-500 μL of 0.2 mg / mL iron-copper doped carbon dot Cu,Fe-CDs solution, 1.0-1.5 mL of 2.2 mg / mL AgNO3 solution, and 100-200 μL of 50 mg / mL lysozyme to 20-30 mL of deionized water, stir at room temperature for 20-30 min, and vacuum dry to obtain Cu,Fe-Lyz / AgNPs nanozyme.

2. The Cu,Fe-Lyz / AgNPs nanozyme prepared by the near-infrared light-enhanced responsive Cu,Fe-Lyz / AgNPs nanozyme preparation method according to claim 1.

3. The application of the Cu,Fe-Lyz / AgNPs nanozyme according to claim 2 in the detection of potassium penicillin G.

4. The application according to claim 3, characterized in that: Cu,Fe-Lyz / AgNPs nanozyme solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 solution were added sequentially to penicillin G potassium solutions of different concentrations. After being diluted to volume with pH 4.0 acetate buffer, the mixture was reacted at room temperature for 10-20 min, and the absorbance was measured at a wavelength of 654 nm to determine the linear relationship between penicillin G potassium concentration and absorbance value, and to obtain the regression equation. Cu,Fe-Lyz / AgNPs nanozyme solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 solution were added to the sample to be tested. After adjusting the volume with pH 4.0 acetate buffer solution, the mixture was reacted at room temperature for 10-20 min. The absorbance value was measured at a wavelength of 654 nm and substituted into the regression equation to obtain the penicillin G potassium content of the sample to be tested.

5. The application according to claim 4, characterized in that: The concentration of Cu,Fe-Lyz / AgNPs nanozyme solution was 1 mg / mL, and the addition amount was 10-20 μL; the concentration of 3,3',5,5'-tetramethylbenzidine solution was 5 mmol / L, and the addition amount was 20-40 μL; the concentration of H2O2 solution was 50 mmol / L, and the addition amount was 20-40 μL; the concentration of pH 4.0 acetate buffer solution was 0.1 mol / L.

6. The application of the Cu,Fe-Lyz / AgNPs nanozyme according to claim 2 in the preparation of antibacterial agents.

7. The application according to claim 6, characterized in that: Cu,Fe-Lyz / AgNPs nanozymes exhibit antibacterial activity in the presence of peroxides and under near-infrared light irradiation.

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