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

Abstract

The application discloses a preparation method of near-infrared light enhanced response Cu, Fe-Lyz / AgNPs nanoscale enzyme. The method uses Cu, Fe doped carbon dots as a reducing agent to prepare silver nanoparticles, and then the silver nanoparticles are modified by lysozyme to prepare Cu, Fe-Lyz / AgNPs nanoscale enzyme with positive charges on the surface. The Cu, Fe-Lyz / AgNPs nanoscale enzyme has dual activities of peroxidase-like and glutathione oxidase-like and photo-thermal performance. Meanwhile, the near-infrared light can significantly enhance the POD-like activity of the nanoscale enzyme. The Cu, Fe-Lyz / AgNPs nanoscale enzyme has good antibacterial effect on escherichia coli and staphylococcus aureus. Meanwhile, based on the enhancement of the penicillin G potassium on the Cu, Fe-Lyz / AgNPs oxidase-like activity, the application establishes a rapid colorimetric detection method for the penicillin G potassium, and when the method is used for detecting the penicillin G potassium in blood 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.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to a method for preparing near-infrared light-enhanced responsive Cu,Fe-Lyz / AgNPs nanozymes and their application in detecting penicillin G potassium and preparing antibacterial agents. Background Technology

[0002] The rising incidence of bacterial infections has become a global concern, threatening human health and life. Even more worrying is the increasing antibiotic resistance due to overuse, while traditional antibiotics are becoming increasingly ineffective. Therefore, developing novel and effective antibacterial agents is crucial. Among various antibacterial materials, carbon dots (CDs) have attracted widespread attention as a novel alternative to traditional antibacterial agents due to their simple synthesis, microbial adhesion, low toxicity, and good biocompatibility. Furthermore, CDs possess enzyme-mimicking activity, generating toxic reactive oxygen species (ROS). Metal doping can enhance ROS production, achieving effective antibacterial effects. Simultaneously, CDs exhibit excellent optical properties, especially metal-doped CDs, which respond to near-infrared (NIR) light, producing a unique photothermal / photodynamic synergistic effect. This provides a broader application scope for the antibacterial use of these materials. The materials can effectively utilize the deep tissue penetration capability of NIR light to achieve precise and controllable bactericidal effects while significantly reducing damage to normal tissues. In addition, NIR-responsive materials have shown significant advantages in anti-infective therapy, such as highly efficient sterilization and reduced drug resistance, bringing revolutionary new ideas to the field of anti-infective therapy.

[0003] Penicillin G potassium (PGK), a broad-spectrum β-lactam antibiotic, is widely used to treat bacterial infections in humans and animals. However, there is a strong awareness that the overuse of antibiotics poses significant risks to ecosystems and human health, as unmetabolized antibiotics can enter water bodies or soil, causing environmental pollution and raising increasing concerns about antibiotic resistance. On February 27, 2017, the World Health Organization (WHO) listed β-lactam-resistant bacteria as one of the most dangerous groups in its list of "12 Most Dangerous Drug-Resistant Bacteria." Since PGK is one of the most frequently detected β-lactam antibiotics in the environment, effective methods are needed for the sensitive detection of PGK in environmental samples. Summary of the Invention

[0004] This invention provides a method for preparing near-infrared light-enhanced responsive Cu,Fe-Lyz / AgNPs nanozymes and their application in detecting penicillin G potassium and preparing antibacterial agents. This invention utilizes 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. The Cu,Fe-Lyz / AgNPs nanozymes possess dual activities of peroxidase-like (POD-like) and glutathione oxidase-like (GSH oxidase-like) properties, as well as photothermal properties. Furthermore, near-infrared light (NIR) significantly enhances the POD-like activity of the nanozymes. The results showed that Cu,Fe-Lyz / AgNPs nanozymes exhibited good antibacterial effects against *Escherichia coli* and *Staphylococcus aureus*. After 7 minutes of treatment with Cu,Fe-Lyz / AgNPs nanozymes followed by NIR irradiation, nearly 100% inactivation of the pathogens and over 90% biofilm inhibition were achieved, with minimum inhibitory concentrations (MICs) of 5 μg / mL and 10 μg / mL, respectively. These effects were attributed to the generation of reactive oxygen species, release of silver ions, depletion of glutathione (GSH), and the positive surface charge of the Cu,Fe-Lyz / AgNPs nanozymes. Furthermore, based on the enhancing effect of potassium penicillin G on the oxidase-like activity of Cu,Fe-Lyz / AgNPs nanozymes, a rapid colorimetric detection of potassium penicillin G was developed. When the established method was applied to the detection of potassium penicillin G in plasma and milk powder samples, the detection limit was as low as 0.05 μg / mL, demonstrating high reliability and accuracy.

[0005] The preparation method of the near-infrared light-enhanced responsive Cu,Fe-Lyz / AgNPs nanozyme of the present invention is as follows:

[0006] (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 0.22μm filter membrane, and vacuum dry to obtain iron-copper doped carbon dots Cu,Fe-CDs;

[0007] The centrifugation is performed at 8000-10000 r / min for 5-10 min;

[0008] (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.

[0009] The Cu,Fe-Lyz / AgNPs nanozyme prepared by the above method was applied to the detection of potassium penicillin G. Specifically, Cu,Fe-Lyz / AgNPs nanozyme solution, 3,3',5,5'-tetramethylbenzidine (TMB) solution, and H2O2 solution were added sequentially to potassium penicillin G solutions of different concentrations. After adjusting the volume with pH 4.0 acetate buffer, the reaction was carried out at room temperature for 10-20 min, and the absorbance value was measured at a wavelength of 654 nm to determine the linear relationship between the concentration of potassium penicillin G and the absorbance value, and the regression equation was obtained.

[0010] 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.

[0011] The Cu,Fe-Lyz / AgNPs nanozyme solution had a concentration of 1 mg / mL and was added in an amount of 10-20 μL; the 3,3',5,5'-tetramethylbenzidine solution had a concentration of 5 mmol / L and was added in an amount of 20-40 μL; the H2O2 solution had a concentration of 50 mmol / L and was added in an amount of 20-40 μL; and the pH 4.0 acetate buffer solution had a concentration of 0.1 mol / L.

[0012] The Cu,Fe-Lyz / AgNPs nanozymes prepared by the above method are used in the preparation of antibacterial agents. The Cu,Fe-Lyz / AgNPs nanozymes achieve antibacterial effects in the presence of peroxides and under near-infrared light irradiation. The Cu,Fe-Lyz / AgNPs nanozymes are used to remove Escherichia coli, Staphylococcus aureus and their biofilms.

[0013] The power of the 808nm near-infrared light is 0.50-1.0W / cm². 2 .

[0014] Advantages and technical effects of the present invention:

[0015] 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;

[0016] 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 The biofilm decreased to about 10%; this "capture-catalysis-kill" three-modal synergistic system provides a new model for developing smart nanomaterials that resist microbial resistance;

[0017] 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.

[0018] 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

[0019] 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.

[0020] Figure 2 The Cu 2p high-resolution XPS spectrum of the Cu,Fe-Lyz / AgNPs nanozyme prepared in Example 1 of this invention;

[0021] Figure 3 The high-resolution XPS spectrum of Fe 2p of the Cu,Fe-Lyz / AgNPs nanozyme prepared in Example 1 of this invention;

[0022] 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.

[0023] Figure 5 The UV-Vis absorption spectra of Cu,Fe-Lyz / AgNPs nanozymes and Cu,Fe-Lyz / AgNPs nanozymes + NIR are shown.

[0024] 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.

[0025] 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.

[0026] 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;

[0027] 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.

[0028] 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.

[0029] Figure 11Figure a shows the GSH oxidase-like enzyme activity detection results 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).

[0030] Figure 12 The effect of different concentrations of Cu,Fe-Lyz / AgNPs nanozymes on the survival rate of Hep G2 cells (Figure a) and HUVECs cells (Figure b) at 24 h and 48 h is shown in the figure. The MIC in the figure is 10 μg / mL.

[0031] Figure 13 Figure a shows the antibacterial performance of different experimental groups against different pathogens under 808nm irradiation and no irradiation (Figure a), and the effect of different concentrations of Cu,Fe-Lyz / AgNPs nanozymes on the antibacterial performance of different pathogens under 808nm irradiation and no irradiation (Figure b).

[0032] Figure 14 For different concentrations of Cu,Fe-Lyz / AgNPs nanozymes E. coli (Figure a) and S.aureus (Figure b) Statistical results of antibacterial performance;

[0033] Figure 15 Inhibition of Cu,Fe-Lyz / AgNPs nanozymes at different concentrations E. coli and S.aureus CV staining results of biofilms;

[0034] Figure 16 Inhibition of Cu,Fe-Lyz / AgNPs nanozymes at different concentrations E. coli (Figure a) and S.aureus (Figure b) shows the statistical results of the biofilm inhibition rate;

[0035] Figure 17 SEM images of different bacteria treated with Cu,Fe-Lyz / AgNPs nanozymes;

[0036] Figure 18 The UV-Vis spectra of H2O2+Cu,Fe-Lyz / AgNPs and H2O2+PGK+Cu,Fe-Lyz / AgNPs are shown.

[0037] Figure 19 The diagram shows the hydroxyl radical capture of H2O2+Cu,Fe-Lyz / AgNPs and H2O2+PGK+Cu,Fe-Lyz / AgNPs.

[0038] Figure 20 Figure 1 shows the UV-Vis absorption spectrum of PGK (Figure 2) and the linear regression equation (Figure 3).

[0039] Figure 21 The results show the influence of coexisting substances on the determination of PGK.

[0040] Figure 22 The results show the effect of metal ions on the determination of PGK. Detailed Implementation

[0041] The following examples further illustrate the content of the present invention, but these examples do not limit the scope of protection of the present invention. Unless otherwise specified, the methods in the examples are conventional methods, and unless otherwise specified, the reagents used are conventional commercial reagents or reagents prepared according to conventional methods.

[0042] Example 1: Preparation and antibacterial detection of Cu,Fe-Lyz / AgNPs nanozymes

[0043] 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 μm filter membrane, and vacuum dried to obtain iron and copper doped carbon dots Cu,Fe-CDs;

[0044] 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.

[0045] 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.

[0046] 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 2In 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 eV) and Fe 3+ The satellite peak (724.5 eV);

[0047] 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. coli , Cu,Fe / AgNPs, Cu,Fe-Lyz / AgNPs and Cu,Fe-Lyz / AgNPs+ E. coli The Zeta values ​​were -11.3 mV, -9.5 mV, 13.1 mV, and 2.6 mV, respectively, indicating that the Cu,Fe-Lyz / AgNPs nanozyme prepared by lysozyme modification has a positively charged surface, and E. coli It can bind to Cu,Fe-Lyz / AgNPs nanozymes;

[0048] 3. Evaluation of the photothermal performance of Cu,Fe-Lyz / AgNPs nanozymes: To evaluate the NIR-triggered photothermal performance of Cu,Fe-Lyz / AgNPs nanozymes, the temperature change of the nanozyme solution under NIR laser irradiation at 808 nm was measured. Characterization by UV-Vis-NIR absorption spectroscopy revealed that the Cu,Fe-Lyz / AgNPs nanozymes exhibited a significant absorption peak at 808 nm. Figure 5 This characteristic peak is directly related to its photothermal energy conversion performance.

[0049] Photothermal heating curves confirmed that the temperature increase was related to the concentration and infrared intensity of the Cu,Fe-Lyz / AgNPs nanozyme. Figure 6 Under irradiation with light at a wavelength of 808 nm, the temperature of a Cu,Fe-Lyz / AgNPs nanozyme solution with a concentration of 200 µg / mL rose to 66.6 °C. This temperature range can effectively induce damage to the bacterial membrane structure. In contrast, the temperature rise of pure water (Blank) under 808 nm infrared irradiation is negligible. The results indicate that Cu,Fe-Lyz / AgNPs nanozymes have the ability to convert NIR light energy into heat energy.

[0050] To provide a foundation for further research on the photothermal performance of Cu,Fe-Lyz / AgNPs nanozymes, the photothermal conversion efficiency (η) of the Cu,Fe-Lyz / AgNPs nanozyme solution (150 μg / mL) was calculated using the following formula. T ):

[0051] ;

[0052] In the formula 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:

[0053] ;

[0054] In the formula 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 the Cu,Fe-Lyz / AgNPs nanozyme solution (150 μg / mL) was obtained as 59.6%. Figure 7 It exhibits a high photothermal conversion efficiency.

[0055] 4. Evaluation of peroxidase-like activity of Cu,Fe-Lyz / AgNPs nanozymes: The peroxidase-like (POD-like) activity was evaluated using TMB as the catalytic reaction substrate. 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 nanozyme solution were added to a 5 mL stoppered colorimetric tube. Then, 0.1 mol / L HAc-NaAc buffer solution (pH 4.0) was added to bring the volume to 3 mL. After thorough mixing and reacting for 10 min, the absorbance was measured at 654 nm.

[0056] Meanwhile, under the same conditions, the reaction system at 1.0 W / cm2 Enzyme activity was compared by irradiation at 808 nm for 7 min, and absorbance was measured at 654 nm. Controls were provided for enzymes with only TMB, only H2O2, and only TMB + H2O2. Results are as follows: Figure 8 When Cu,Fe-Lyz / AgNPs nanozymes and H2O2 were present in the system, TMB underwent a significant oxidation reaction. This experimental phenomenon fully demonstrates that Cu,Fe-Lyz / AgNPs nanozymes possess POD-like catalytic activity and can mimic the function of natural peroxidases, catalyzing the oxidation of TMB by H2O2 to generate a blue product. Furthermore, after applying NIR irradiation to the reaction system for 7 min, the POD-like activity of Cu,Fe-Lyz / AgNPs nanozymes was significantly enhanced, with an increase of nearly 4 times. Cu,Fe-Lyz / AgNPs nanozymes exhibit the characteristic of near-infrared light-enhanced peroxidase-like activity.

[0057] The experiment also included the determination of Michaelis catalytic kinetic parameters. Figure 9 , Figure 10 (and Table 1), Michaelis constants of Cu,Fe-Lyz / AgNPs nanozymes for substrates TMB and H2O2. K m The reaction rate constants were 3.88 × 10⁻⁴ mM and 0.20 mM, respectively. -8 M / s and 1.20×10 -8 M / s, after irradiation with 808nm infrared light K m The reaction rate constants are 22.4 × 10⁻⁶ for 0.23 mM and 0.18 mM. -8 M / s and 1.58×10 -8 M / s indicates that infrared light irradiation increased the affinity and reaction rate between Cu,Fe-Lyz / AgNPs nanozymes and substrates;

[0058] Table 1 Michaelis catalytic kinetic parameters

[0059] ;

[0060] 5. Evaluation of glutathione oxidase (GSH oxidase-like) activity: Add 50 μL of 2 mg / mL Cu,Fe-Lyz / AgNPs nanozyme solution to 100 μL of a mixed solution of 100 mM 5,5′-dithiodinitrophenol (DTNB) and 1 mL of 2 mg / mL GSH, and dilute to 5 mL with water. Then observe the change in absorbance at 420 nm. The results are as follows: Figure 11As shown in Figure a, compared with the blank group, the absorbance value of the reaction system with added nanozymes decreased significantly at 420 nm, indicating that Cu,Fe-Lyz / AgNPs have significant GSH scavenging activity. Furthermore, as... Figure 11 As shown in b, under NIR irradiation, by testing Cu,Fe-Lyz / AgNPs nanozyme solutions of different concentrations, it was found that the GSH oxidase-like enzyme activity increased with increasing concentration, indicating that the enzyme activity was positively correlated with concentration. All of the above results indicate that Cu,Fe-Lyz / AgNPs nanozymes can effectively oxidize GSH to oxidized glutathione (GSSG).

[0061] 6. Cytotoxicity Assay: The cytotoxicity of Cu,Fe-Lyz / AgNPs nanozymes was detected using a CCK-8 cell viability assay kit. Human umbilical vein endothelial cells and liver cancer cells (HUVECs and HepG2, Beina Chuanglian Biotechnology Co., Ltd.) were seeded into 96-well plates and cultured for 24 h. After incubation with different concentrations of Cu,Fe-Lyz / AgNPs nanozyme solution for 24 h and 48 h, the cells were washed with PBS, and CCK-8 solution was added to each well to a concentration of 10%. The cells were incubated at 37 °C, and the absorbance was measured at 450 nm. CCK-8 analysis (…) Figure 12 The results showed that Cu,Fe-Lyz / AgNPs nanozymes were not toxic to cells.

[0062] 7. Antibacterial experiment of Cu,Fe-Lyz / AgNP nanozyme

[0063] The following bacterial strains were obtained from Beina Innovation Biotechnology Co., Ltd.

[0064] 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.

[0065] 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.

[0066] Figure 13 b、 Figure 14 As shown, in the presence of H2O2, Cu,Fe-Lyz / AgNPs nanozymes exhibit significant concentration-dependent antibacterial effects against different microorganisms; and against Gram-negative bacteria... E. coli When the concentration of Cu,Fe-Lyz / AgNPs is 5 μg / mL, the sterilization rate is 99.9%; against Gram-positive bacteria S. aureus It achieved an inhibition rate of 99.8% at a concentration of 10 μg / mL.

[0067] To further evaluate the anti-biofilm performance of Cu,Fe-Lyz / AgNPs nanozymes, we investigated their anti-biofilm ability using crystal violet (CV) staining and quantified biofilm activity by measuring absorbance at 590 nm. Different concentrations of Cu,Fe-Lyz / AgNPs nanozymes, H2O2 (0.5 mmol / L), and... E. coli or S.aureus After mixing and irradiating with 808nm for 30 min, the mixture was placed in a constant temperature incubator and incubated for 24 h. The bacterial biofilm production rate was then compared with that of the blank control group, the group without infrared irradiation, and the control group with only H2O2 added. Figure 15 , 16The results showed that in the presence of H2O2, increasing the content of Cu,Fe-Lyz / AgNPs nanozymes led to a decrease in biofilm formation; 100 μg / mL Cu,Fe-Lyz / AgNPs could reduce biofilm formation. E. coli The biofilm decreased to approximately 7.13%. S.aureus The concentration was 11.75%, indicating that Cu,Fe-Lyz / AgNPs have excellent inhibitory ability on biofilm formation; Cu,Fe-Lyz / AgNPs+NIR nanozymes have... E. coli and S.aureus The biofilms were all inhibited, and the Cu,Fe-Lyz / AgNPs+NIR effect was better than that of Cu,Fe-Lyz / AgNPs nanozymes without irradiation.

[0068] Figure 17 Scanning electron microscopy (SEM) characterization revealed that the control group bacteria exhibited typical rod-shaped or spherical ultrastructures, maintaining cell membrane integrity and confirming that they were not affected by the nanomaterials. After treatment with Cu,Fe-Lyz / AgNPs + H₂O₂ + NIR, the bacteria showed characteristic structural damage: ① membrane surface wrinkling; ② cell collapse; ③ intracellular leakage. This multi-level structural damage mode is closely related to the cascade catalytic properties of the nanozyme—the enhanced electron transfer efficiency of NIR increased the quantum yield of H₂O₂ to ·OH, thereby triggering a cascade reaction of bacterial membrane lipid peroxidation, revealing the antibacterial pathway of the material.

[0069] The above results indicate that the Cu,Fe-Lyz / AgNPs nanozymes prepared in this invention exhibit high peroxidase activity and photothermal properties under NIR irradiation, catalyzing the production of ROS with bactericidal effects from H2O2, thereby enhancing the peroxidase activity of H2O2. E. coli and S. aureus It exhibited good antibacterial activity. Photoresponse characterization showed that this nanozyme system displayed a dual-modal antibacterial mechanism under 808nm NIR excitation: ① increased POD-like activity; ② photothermal conversion efficiency η=59.6%, enabling localized temperature rise. It also showed activity against Gram-negative bacteria. E. coli When the concentration of Cu,Fe-Lyz / AgNPs is 5 μg / mL, the sterilization rate is 99.9%; against Gram-positive bacteria S. aureus It achieved an inhibition rate of 99.8% at a concentration of 10 μg / mL.

[0070] 9. Cu,Fe-Lyz / AgNPs nanozyme for colorimetric detection of potassium penicillin G (PGK)

[0071] (1) Effect of PGK on the POD-like activity of Cu,Fe-Lyz / AgNPs nanozyme: 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 nanozyme, 50 μL of 10 μg / mL PGK solution, and 200 μL of 0.1 mol / L pH 4.0 HAc-NaAc buffer solution were added to a 96-well plate. After mixing, the mixture was reacted at room temperature for 10 min, and the absorbance was measured at 654 nm. A control group without PGK was used. The results are as follows: Figure 18 PGK significantly increases POD-like activity.

[0072] (2) Detection of hydroxyl radicals (·OH): Terephthalic acid (TA) was used as a probe to detect ·OH. TA is oxidized to 2-hydroxyterephthalic acid (TAOH) in the presence of ·OH. 100 μL of 1 mg / mL Cu,Fe-Lyz / AgNPs nanozyme, 100 μL of 5 mg / mL TA, and 200 μL of 50 mmol / L H2O2 were added to 2.6 mL of pH 4.0 HAc-NaAc buffer solution, mixed thoroughly, and incubated at room temperature for 4 h. The fluorescence was measured at 438 nm under an excitation wavelength of 330 nm to investigate its catalytic activity. The results are shown in […]. Figure 19 Cu,Fe-Lyz / AgNPs+H2O2 has a strong ability to generate ·OH. Under the same detection conditions, the addition of PGK in the experiment showed that the addition of PGK increased the fluorescence intensity of the system, indicating that the addition of PGK is conducive to the generation of ·OH. This confirms that PGK improves POD-like activity by generating more ·OH.

[0073] (3) Preparation of PGK working curve: 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 nanozyme, 50 μL of PGK solutions of different concentrations, and 200 μL of 0.1 mol / L pH 4.0 HAc-NaAc buffer solution to a 96-well plate, mix well, react at room temperature for 10 min, and measure the absorbance at 654 nm. Simultaneously, set up a blank control without PGK, and calculate ΔA = A - A0, where A0 is the absorbance of the blank control and A is the absorbance with added PGK. Plot the calibration curve of absorbance difference ΔA versus PGK concentration, see [reference needed]. Figure 20 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are obtained and are shown in Table 2.

[0074] Table 2. Linear equation, correlation coefficient, relative standard deviation, and linear range

[0075]

[0076] (4) Method specificity study: PGK was replaced 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), roxithromycin (PNZ)), and coexisting ions (Na+). + K + Ca 2+ Mg 2+ Cu 2+ Zn 2+ Fe 2+ Cl - SO4 2- NO3 - The effects of PGK and other substances on the Cu,Fe-Lyz / AgNPs nanozyme in step (3) were detected; the concentrations of PGK and other antibiotics were 5 μg / mL, and the concentration of coexisting ions was 50 μg / mL. The results are shown in […]. Figure 21 , 22 As can be seen from the figure, the Cu,Fe-Lyz / AgNPs nanozyme detection system has good selectivity and specificity. PGK significantly enhances the oxidation reaction, while other substances are almost absent. The method of this invention has good selectivity and specificity for the determination of PGK.

[0077] (5) Determination of PGK in the sample

[0078] Milk powder sample: First, dissolve 2.5g of milk powder in 20mL of water at 25℃, then add 8.0mL of acetic acid (3%, v / v), stir vigorously for 15min, dilute with 50mL of deionized water, centrifuge at 4000rpm for 15min, and take the supernatant to obtain the test solution;

[0079] Plasma samples: taken from Kunming University of Science and Technology Hospital, diluted 50 times with pH 7.0 phosphate buffer (PBS, 0.1 mol / L) solution to obtain the test solution;

[0080] PGK standard solutions of 0.5, 10, and 50 μg / mL were added to the above test solution, and the spiked recovery rate test was carried out on the test sample. The results are shown in Table 3. The results show that the PGK spiked recovery rate in the test sample is between 93.9% and 106.1%, and the RSD is less than 4%. The results indicate that the method established in this invention can be used for the detection of PGK in milk powder and plasma samples.

[0081] Table 3. Spike recoveries and RSDs of samples (n=3)

[0082]

[0083] The PGK determination method established in this invention has advantages in practical testing because it involves fewer processing steps, shorter processing time, lower processing cost, simpler operation, and does not require large-scale instruments and equipment.

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 0.22μm 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. The absorbance value was then 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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