Porous palladium nanoflower as well as preparation method and application thereof

Abstract

The invention discloses a porous palladium nanoflower, a preparation method thereof and application of the porous palladium nanoflower in efficient bacteriostasis, and belongs to the field of porous materials. According to the preparation method, a carbon quantum dot mediation method is utilized, carbon quantum dots and a palladium source are mixed, and a reducing agent is added for reaction, so that the porous palladium nanoflower is prepared. The obtained porous palladium nanoflower is formed by gathering palladium nanocrystals with the particle size of 6.2-7.4 nm, the surface is rough, and the porous structure is achieved, so that the oxidase-like activity of the porous palladium nanoflower is improved, the adhesion of bacteria can be enhanced, the antibacterial efficiency is improved, and the use amount of nano materials is reduced.

Description

Technical Field

[0001] The invention belongs to the field of nanomaterials, and specifically relates to a preparation method of porous palladium nanoflowers and application of the porous palladium nanoflowers in the preparation of antibacterial agents. Background Art

[0002] Infectious diseases caused by bacteria are posing a serious threat to public health. Due to the increase in bacterial resistance, there is an urgent need to develop a new type of antimicrobial agent. Killing bacteria through reactive oxygen species (ROS) is considered an effective strategy to address bacterial antibiotic resistance. In the past decade, nanostructures with oxidase-like (OXD) activity have attracted considerable research interest in antimicrobial drug research because they are able to produce a lethal ROS storm without the help of hydrogen peroxide. However, at present, the catalytic efficiency of OXD nanozymes is limited, resulting in a low yield of ROS. On the other hand, the inherent disadvantages of ROS, such as short lifespan, limited range of action, and the need for a large number of nanozymes to achieve effective bacterial inhibition, also seriously affect the bactericidal effect of these materials and will lead to potential risks. Therefore, finding new materials that can simultaneously improve the OXD catalytic efficiency of nanozymes and improve the interaction between nanozymes and bacteria is the preferred strategy.

[0003] Generally speaking, the smaller the size of the nanoparticles, the better the catalytic performance and antibacterial activity. However, smaller particles are more likely to aggregate, which will lead to a direct reduction in surface active sites, thereby limiting the catalytic and antibacterial activity. The porous nanoflowers formed by the assembly of nanocrystals can not only avoid the inactivation caused by agglomeration, but also can retain the catalytic surface of the nanocrystals to the maximum extent, and have been proven to be an effective means to obtain efficient catalytic activity. On the other hand, rough surfaces have been shown to increase bacterial adhesion, thereby promoting antibacterial activity. Based on this, the present invention adopts a CDs-mediated method to develop a porous palladium nanoflower with good OXD mimetic activity that can be used for antibacterial purposes. Summary of the invention

[0004] The purpose of the present invention is to provide a porous palladium nanoflower and its preparation method and application. The porous palladium nanoflower prepared by the method of the present invention is like an aggregate of highly active palladium nanocrystals, has a high oxidase-like catalytic efficiency, and its surface is rough, which is conducive to the adhesion of bacteria, thereby improving its antibacterial efficiency and reducing the use of nanomaterials.

[0005] To achieve the above object, the present invention adopts the following technical solution: The first objective of the present invention is to protect a method for preparing a porous palladium nanoflower, which comprises mixing carbon quantum dots and a palladium source evenly in water, adding a reducing agent to react, and thus preparing the porous palladium nanoflower.

[0006] Furthermore, the particle size of the carbon quantum dots is less than 5 nm.

[0007] Furthermore, the preparation method of the carbon quantum dots includes one or more of a microwave method, an electrochemical method, a glucose cleavage method, or other existing methods.

[0008] Furthermore, the palladium source includes one or more of palladium chloride, potassium chloropalladate, and chloropalladic acid.

[0009] Furthermore, in the mixed reaction solution, the molar concentration of palladium atoms is 0.1-5 mmol / mL, and the mass concentration of carbon quantum dots is 10-2000 μg / mL.

[0010] Furthermore, the reducing agent is ascorbic acid.

[0011] Furthermore, the molar ratio of the reducing agent to the palladium atom is 6:1.

[0012] Furthermore, the reaction temperature is 30-40°C and the reaction time is 0.5-4h.

[0013] Furthermore, the obtained porous palladium nanoflowers are formed of palladium nanocrystals with a particle size of about 6.2nm~7.4nm, and the particle size is above 35.8nm.

[0014] The second object of the present invention is to protect the porous palladium nanoflowers prepared by the above method.

[0015] The third objective of the present invention is to protect the application of the porous palladium nanoflowers as oxidase-like enzymes, and the oxidase-like enzyme activity of the porous palladium nanoflowers can increase as the particle size thereof increases.

[0016] The fourth object of the present invention is to protect the use of the porous palladium nanoflowers as an antibacterial agent.

[0017] Compared with the prior art, the present invention has the following significant advantages: (1) The present invention provides a method for preparing a porous palladium nanomaterial. The porous palladium nanomaterial is formed by aggregation of small-sized palladium nanocrystals under the mediation of carbon quantum dots. During the aggregation process of the palladium nanocrystals, the carbon quantum dots are expelled and a porous structure is formed between the crystals. Therefore, the good catalytic activity of the small-sized palladium nanocrystals can be well retained. At the same time, the rough structure on the surface of the porous palladium nanomaterial can increase the adhesion of bacteria.

[0018] (2) The larger the particles of the porous palladium nanoflowers obtained by the present invention, the better the simulated oxidase activity and the higher the catalytic efficiency. The activity decline phenomenon caused by the agglomeration of small particles is avoided, and the nanoflowers have good stability. The highly efficient oxidase-like activity can convert dissolved oxygen into more toxic ROS in a short time, forming a ROS storm, thereby achieving a highly efficient sterilization effect. The Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus were used as models to study its antibacterial activity against bacteria. The results showed that the half-inhibitory concentrations of porous palladium nanoflowers (Pd NDs-1) with a particle size of about 35.8 nm, the porous palladium nanoflowers (Pd NDs-2) with a particle size of about 58.9 nm, and the cloud-shaped porous palladium nanoflowers (PdNDs-3) with a particle size greater than 100 nm against Escherichia coli were 5.8, 8.7, and 2.0 μg / mL, respectively, and the half-inhibitory concentrations against Staphylococcus aureus were 6.2, 6.3, and 2.2 μg / mL, respectively; Pichia pastoris was selected as a model to study its antibacterial activity against fungi. The results showed that the half-inhibitory concentration of Pd NDs-3 against Pichia pastoris was 11.4 μg / mL.

[0019] (3) The method provided by the present invention has mild and simple reaction conditions. The obtained porous palladium nanomaterial is an aggregate of palladium nanodendrites. The larger the particles, the higher the simulated oxidase activity and the higher the catalytic efficiency. In addition, its structure is conducive to the adhesion of bacteria, which can greatly improve the antibacterial activity and effectively reduce the amount of nanomaterials used. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 TEM image and particle size distribution diagram of the porous palladium nanoflowers Pd NDs-1 prepared in Example 1.

[0021] Figure 2 This is a high-resolution electron microscope image of the porous palladium nanoflowers Pd NDs-1 prepared in Example 1.

[0022] Figure 3 This is the particle size distribution diagram of palladium nanocrystals in the porous palladium nanoflower Pd NDs-1 prepared in Example 1.

[0023] Figure 4 TEM image and particle size distribution diagram of the porous palladium nanoflowers Pd NDs-2 prepared in Example 2.

[0024] Figure 5 This is a high-resolution electron microscope image of the porous palladium nanoflowers Pd NDs-2 prepared in Example 2.

[0025] Figure 6 This is the particle size distribution diagram of palladium nanocrystals in the porous palladium nanoflowers Pd NDs-2 prepared in Example 2.

[0026] Figure 7TEM image of the porous palladium nanoflowers Pd NDs-3 prepared in Example 3.

[0027] Figure 8 This is a high-resolution electron microscope image of the porous palladium nanoflowers Pd NDs-3 prepared in Example 3.

[0028] Fig. 9 This is a particle size distribution diagram of palladium nanocrystals in the porous palladium nanoflower Pd NDs-3 prepared in Example 3; Fig.10 This is the XRD pattern of the porous palladium nanoflowers prepared in Example 1-3.

[0029] Fig.11 This is the nitrogen adsorption-desorption isotherm diagram of the porous palladium nanoflowers prepared in Example 1-3.

[0030] Fig.12 This is the TEM image of the palladium nanomaterial Pd NPs prepared in Comparative Example 1.

[0031] Fig.13 The kinetic curves of TMB oxidation catalyzed by the porous palladium nanoflowers prepared in the example and the palladium nanomaterials prepared in the comparative example.

[0032] Fig.14 This is the steady-state kinetic curve of TMB oxidation catalyzed by the porous palladium nanoflowers prepared in Example.

[0033] Fig.15 It is a flat plate experiment diagram after the porous palladium nanoflowers prepared in the example and the palladium nanomaterials prepared in the comparative example were co-incubated with Gram-negative Escherichia coli or Gram-positive Staphylococcus aureus.

[0034] Fig.16 This is a flat plate experiment diagram after the porous palladium nanoflowers Pd NDs-3 prepared in Example 3 were co-incubated with Pichia pastoris.

[0035] Fig.17 This is a live-dead fluorescent staining image of Staphylococcus aureus after the porous palladium nanoflowers Pd NDs-3 prepared in Example 3 were co-incubated with Staphylococcus aureus.

[0036] Fig.18 This is a live-dead fluorescent staining image of Escherichia coli after the porous palladium nanoflowers Pd NDs-3 prepared in Example 3 were co-incubated with Escherichia coli.

[0037] Fig.19 This is a scanning electron microscope image (high resolution) of the porous palladium nanoflowers prepared in Example after incubation with Gram-negative Escherichia coli or Gram-positive Staphylococcus aureus.

[0038] Fig. 20This is a scanning electron microscope image (high resolution) of the porous palladium nanoflowers Pd NDs-3 prepared in Example 3 after incubation with Pichia pastoris.

[0039] Fig.21 This is a fluorescence imaging image of ROS in cells after the porous palladium nanoflowers Pd NDs-3 prepared in Example 3 were co-incubated with Staphylococcus aureus, Escherichia coli and Pichia pastoris. DETAILED DESCRIPTION

[0040] The invention provides a method for preparing porous palladium nanoflowers, which comprises the steps of uniformly mixing carbon quantum dots and a palladium source in water, and then adding a reducing agent to react to prepare the nanoflowers.

[0041] In the present invention, unless otherwise specified, the raw materials used are conventional commercial products in the art.

[0042] In the present invention, the palladium source, water and carbon quantum dots are mixed and then a reducing agent is added to react. Preferably, the palladium source and water are first mixed to prepare a palladium source solution; then the palladium source solution and carbon quantum dots are mixed, and then mixed with the reducing agent.

[0043] In the present invention, the carbon quantum dots are preferably added in the form of a carbon quantum dots aqueous solution.

[0044] In the present invention, the carbon quantum dots are one or more carbon quantum dots with a particle size less than 5 nm, which can be prepared by microwave method, electrochemical method, glucose cleavage method, or other existing methods.

[0045] In the present invention, if the carbon quantum dots are prepared by glucose cleavage method, the mass concentration of the carbon quantum dots in the mixed reaction solution is preferably 10-2000 μg / mL, more preferably 2000 μg / mL.

[0046] In the present invention, if the carbon quantum dots are prepared by a microwave method, the mass concentration of the carbon quantum dots in the mixed reaction solution is preferably 28.5-228 μg / mL, more preferably 170 μg / mL.

[0047] In the present invention, if the carbon quantum dots are prepared by cracking a mixture of glucose and urea, the mass concentration of the carbon quantum dots in the mixed reaction solution is preferably 10-1000 μg / mL, more preferably 500 μg / mL.

[0048] In the present invention, the palladium source preferably includes one or more of palladium chloride, potassium chloropalladate and chloropalladic acid. In an embodiment of the present invention, the palladium source is specifically palladium chloride.

[0049] In the present invention, the molar concentration of palladium atoms in the mixed reaction solution is 0.1-5 mmol / mL.

[0050] In the present invention, the reducing agent is preferably ascorbic acid.

[0051] In the present invention, the molar ratio of palladium atoms in the palladium source to the reducing agent is 1:6.

[0052] In the present invention, the reaction is preferably carried out on a shaker with a shaking speed of 220 rpm. The present invention has no particular limitation on the shaker, and any equipment known in the art can be used.

[0053] In the present invention, the reaction temperature is 30-40°C, preferably 37°C.

[0054] In the present invention, if the carbon quantum dots are prepared by glucose cleavage method, the reaction time is 0.5-3h, preferably 2h.

[0055] In the present invention, if the carbon quantum dots are prepared by microwave method, the reaction time is 0.1-4h, preferably 1h.

[0056] In the present invention, if the carbon quantum dots are prepared by cracking a mixture of glucose and urea, the reaction time is 0.5 to 6 hours, preferably 1 hour.

[0057] In the present invention, after the reaction is completed, the reaction product is preferably washed and then dispersed in ultrapure water for later use.

[0058] In the present invention, the solvent used for washing is preferably ultrapure water. The washing method is preferably ultrasonic centrifugal washing. The number of washing times is preferably 3 to 5 times.

[0059] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] Example 1 1920 μL of ultrapure water, 800 μL of 10 mg / mL carbon quantum dot (CDs-1) aqueous solution, 800 μL of 10 mmol / L palladium chloride solution and 480 μL of 100 mmol / L ascorbic acid solution were mixed (the final molar concentration of palladium atoms was 2 mmol / L, the molar concentration of ascorbic acid was 12 mmol / L; the final mass concentration of carbon quantum dots was 2000 μg / mL), and incubated on a shaker (speed of 220 rpm) at a constant temperature of 37°C for 2 h. The product was collected and washed three times by ultrasonic dispersion and centrifugation with ultrapure water. The obtained material was labeled as Pd NDs-1.

[0061] The carbon quantum dots (CDs-1) used were obtained by glucose cleavage and then purified by ultrapure water dialysis, and their particle size was 2.0 nm.

[0062] Figure 1 The TEM image and particle size distribution of the prepared Pd NDs-1. As can be seen from the figure, the prepared porous palladium nanoflower PdNDs-1 is formed by small-sized nanocrystals, the pores between the small-sized nanocrystals are clearly visible, the overall morphology of the particles is flower-like, the surface is rough, and the particle size is about 35.8 nm.

[0063] Figure 2 The high-resolution electron microscope (HRTEM) image of the prepared Pd NDs-1 shows that the particle surface has clearly visible lattice fringes, and the interplanar spacing is 0.25nm, which corresponds to the interplanar spacing of palladium (111).

[0064] Figure 3 The particle size distribution diagram of palladium nanocrystals in the prepared Pd NDs-1. As can be seen from the figure, the particle size of the small palladium nanocrystal particles constituting the porous palladium nanoflower is about 7.4nm.

[0065] Example 2 1920 μL of ultrapure water, 800 μL of 850 μg / mL carbon quantum dot (CDs-2) aqueous solution, 800 μL of 10 mmol / L palladium chloride solution and 480 μL of 100 mmol / L ascorbic acid solution were mixed (the final molar concentration of palladium atoms was 2 mmol / L, the molar concentration of ascorbic acid was 12 mmol / L; the mass concentration of carbon quantum dots was 170 μg / mL), and incubated on a shaker (speed of 220 rpm) at a constant temperature of 37°C for 2 h. The product was collected and washed three times by ultrasonic dispersion and centrifugation with ultrapure water. The obtained material was labeled Pd NDs-2.

[0066] The carbon quantum dots (CDs-2) used were prepared by microwave method and then purified by ultrapure water dialysis, and their particle size was 3.2 nm.

[0067] Figure 4 The TEM image and particle size distribution diagram of the prepared Pd NDs-2. As can be seen from the figure, the prepared Pd NDs-2 is similar to the Pd NDs-1 obtained in Example 1 in morphology, and both are formed by small-sized nanocrystals. The pores between the small-sized nanocrystals are clearly visible, the overall morphology of the particles is flower-shaped, the surface is rough, and the particle size is about 58.9 nm.

[0068] Figure 5The high-resolution electron microscope (HRTEM) image of the prepared Pd NDs-2 shows that the particle surface has clearly visible lattice fringes, and the interplanar spacing is 0.25nm, which corresponds to the interplanar spacing of palladium (111).

[0069] Figure 6 The particle size distribution diagram of palladium nanocrystals in the prepared Pd NDs-2. As can be seen from the figure, the particle size of the small palladium nanocrystal particles constituting the porous palladium nanoflower is about 6.8nm.

[0070] Example 3 1920 μL of ultrapure water, 800 μL of 2500 μg / mL carbon quantum dot (CDs-3) aqueous solution, 800 μL of 10 mmol / L palladium chloride solution and 480 μL of 100 mmol / L ascorbic acid were mixed (the final molar concentration of palladium atoms was 2 mmol / L, the molar concentration of ascorbic acid was 12 mmol / L; the mass concentration of carbon quantum dots was 500 μg / mL), and incubated on a shaker (speed of 220 rpm) at a constant temperature of 37°C for 2 h. The product was collected and washed three times by ultrasonic dispersion and centrifugation with ultrapure water. The obtained material was labeled Pd NDs-3.

[0071] The carbon quantum dots (CDs-3) used were obtained by mixed cracking of glucose and urea and then purified by dialysis with ultrapure water, and their particle size was 3.2 nm.

[0072] Figure 7 The TEM image and particle size distribution of the prepared Pd NDs-3. As can be seen from the figure, the prepared porous palladium nanoflower PdNDs-3 is formed by small-sized nanocrystals, the pores between the small-sized nanocrystals are clearly visible, the overall morphology of the particles is cloud-like, and the surface is rough.

[0073] Figure 8 The high-resolution electron microscope (HRTEM) image of the prepared Pd NDs-3 shows that the particle surface has clearly visible lattice fringes, and the interplanar spacing is 0.25nm, which corresponds to the interplanar spacing of palladium (111).

[0074] Fig. 9 The particle size distribution diagram of palladium nanocrystals in the prepared Pd NDs-3. As can be seen from the figure, the particle size of the small palladium nanocrystal particles that make up the porous palladium nanoflower is about 6.2 nm.

[0075] Fig.10 The XRD diagram of the porous palladium nanoflower prepared in Examples 1 to 3. As can be seen from the figure, the materials prepared in Examples 1 to 3 are all composed of palladium.

[0076] Fig.11The nitrogen adsorption-desorption isotherms of the porous palladium nanoflowers prepared in Examples 1 to 3 are shown in Figure 1. According to the IUPAC classification, the obtained curve is a type IV isotherm, indicating that the obtained materials are all porous materials.

[0077] Comparative Example 1 2720 ​​μL of ultrapure water, 800 μL of 10 mmol / L palladium chloride solution and 480 μL of 100 mmol / L ascorbic acid solution were mixed (the final molar concentration of palladium atoms was 2 mmol / L, and the molar concentration of ascorbic acid was 12 mmol / L), and incubated on a shaker (speed of 220 rpm) at a constant temperature of 37°C for 2 h. The product was collected and washed three times by ultrasonic dispersion and centrifugation with ultrapure water. The obtained material was labeled Pd NPs.

[0078] Fig.12 The TEM image of the prepared Pd NPs shows that, without the participation of carbon quantum dots, the surface of the obtained material is relatively flat, no pores are observed, and the particle size is about 60-70 nm.

[0079] Activity test 1. Comparison of simulated oxidase activity TMB was used as a color developer to detect the oxidase-like activity of the palladium nanomaterials prepared in the examples and comparative examples. The detection principle is: TMB is usually a colorless substance in water, but in the presence of oxidase, the oxidase can oxidize the colorless TMB into a blue substance (the oxidized state of TMB), and there is an obvious absorption peak at 652nm. Therefore, the oxidase-like activity of different nanozymes can be compared by detecting the change of the absorption peak over time. At the same enzyme concentration, the larger the slope of the curve, the stronger the activity of the enzyme.

[0080] The detection process is as follows: the palladium nanomaterials prepared in the embodiment and the comparative example are respectively prepared into 3 mL of 20 mM PB buffer solution (pH 4.0) containing 0.2 mM TMB and 5 μg / mL palladium nanomaterials, the absorbance value at 652 nm is detected as a function of time, and the corresponding curve is plotted.

[0081] Fig.13 The kinetic curves of TMB oxidation catalyzed by the porous palladium nanoflowers prepared in the example and the palladium nanomaterials prepared in the comparative example. As can be seen from the figure, the characteristic absorption peaks of the TMB oxidation state at 652nm obtained by treating different materials gradually increase with time, indicating that these materials have oxidase-like activity. Among them, the degree of oxidation of TMB obtained by treating the porous palladium nanoflowers obtained in the example is significantly deeper, so the slope of its curve is significantly higher than that of the comparative example, proving that the porous palladium nanomaterials prepared in the example have better catalytic efficiency.

[0082] 2. Porous palladium nanoflowers mimic V oxidase max , K m and K cat The detection process is as follows: first, a PB buffer solution (20 mM, pH 4.0) containing different concentrations of TMB (TMB concentrations are 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 150 μM and 200 μM) is prepared, and the porous palladium nanomaterial prepared in the example is added at a certain concentration. After the porous palladium nanoflowers are added, the absorbance value of the system changes with time at 652 nm to obtain the relationship between the initial rate and the TMB concentration, and then the data is fitted by the Lineweaver-Burk double reciprocal equation to obtain V max and K m .

[0083] Lineweaver-Burk double reciprocal equation: ; in, v is the initial velocity, V max is the maximum reaction rate, K m is the Michaelis constant, and [S] is the TMB concentration.

[0084] Fig.14 The steady-state kinetic curve of the porous palladium nanoflowers prepared in Example catalyzing the oxidation of TMB. As can be seen from the figure, the catalytic oxidation of TMB by the porous palladium nanoflowers prepared in Example conforms to the Michaelis equation. The K values ​​of the porous palladium nanoflowers Pd NDs-1, Pd NDs-2 and PdNDs-3 were calculated. m 87.51μM, 48.87μM and 237.4μM respectively, V max 0.08 μM·s -1 , 0.10μM·s -1 and 0.32 μM·s -1 .

[0085] Taking one palladium nanocrystal as one catalytic entity, calculate the molar concentration of nanozyme (C [纳米酶] ), and through K cat =V max / C [纳米酶] , the K of Pd NDs-1, Pd NDs-2 and PdNDs-3 can be calculated cat 26s respectively -1 , 25s -1 and 57s -1Pd NDs-1, Pd NDs-2 and PdNDs-3 are composed of small nanocrystals, so the K of Pd NDs-1 and Pd NDs-2 nanocrystals can be calculated. cat 1.5×10 3 s -1 and 1.5×10 3 s -1 ; The size of Pd NDs-3 is larger than that of Pd NDs-1 and Pd NDs-2, which shows that its K cat The above results show that the prepared porous palladium nanoflowers have good oxidase-like activity, and the larger the particles of the porous palladium nanoflowers, the higher their catalytic efficiency.

[0086] 3. Antibacterial Activity 1.1 Antibacterial activity of porous palladium nanoflowers against Gram-like bacteria Escherichia coli was selected as the representative of Gram-negative bacteria and Staphylococcus aureus was selected as the representative of Gram-positive bacteria. The antibacterial activity of the porous palladium nanoflowers prepared in the example against Gram bacteria was evaluated by using the Gram-antibacterial coating plate and the OD600 absorbance method, and the palladium nanomaterial prepared in the comparative example was used as a control.

[0087] The experimental method is as follows: A. Gram-smear plate: Take three generations of fresh logarithmic growth phase bacteria (OD600≈0.65), centrifuge at 12000 rpm to remove the supernatant culture medium, then wash and centrifuge with the prepared phosphate buffer, then add phosphate buffer to disperse and dilute to a bacterial concentration of 5×10 6 CFU / mL, add different concentrations of palladium nanomaterials, incubate at 37℃ for 1 h, take an appropriate amount of reaction solution to coat the plate, put it upside down in a biochemical incubator, and culture it at 37℃ for 18 h before taking pictures and observing.

[0088] B. Optical density measurement of antibacterial experiment: The reaction solution after the antibacterial incubation experiment was inoculated into the freshly prepared and sterilized culture medium and cultured at 37°C and 220 rpm until the optical density of the blank control group was about 0.65 (5×10 8 CFU / mL), and measured the optical density (n=3).

[0089] like Fig.15As shown in the figure, the growth inhibition effects of different palladium nanomaterials on Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus were dose-dependent, but the porous palladium nanoflowers prepared in the example had more effective antibacterial activity than the Pd NPs prepared in the comparative example, among which Pd NDs-3 had the best antibacterial activity. When the concentration of Pd NDs-3 was 5 μg / mL, the survival rates of Staphylococcus aureus and Escherichia coli were reduced to 0.3 and 0%, respectively.

[0090] Based on the OD600 method, the half-maximal inhibitory concentration (half-inhibitory concentration) values ​​of different porous palladium nanoflowers against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus were determined. The results are shown in Table 1.

[0091] Table 1 IC values ​​of porous palladium nanoflowers against different bacteria 50 value

[0092] As shown in Table 1, Pd NDs-3 has the best antibacterial activity against Staphylococcus aureus and Escherichia coli, with half-inhibitory concentrations of 2.0 μg / mL and 2.2 μg / mL, respectively.

[0093] 1.2. Antibacterial activity of porous palladium nanoflowers against Pichia pastoris Taking Pd NDs-3 prepared in Example 3 as a representative, the antibacterial activity of porous palladium nanoflowers against Pichia pastoris fungi was studied according to the above experimental method.

[0094] like Fig.16 As shown, the inhibitory effect of Pd NDs-3 on the growth of Pichia pastoris was dose-dependent.

[0095] The half-maximal inhibitory concentration (HIC) value of Pd NDs-3 against Pichia pastoris was determined to be 11.4 μg / mL based on the OD600 method.

[0096] 2. Fluorescent live / dead staining was used to further determine the cell death induced by porous Pd nanoflowers.

[0097] Since Pd NDs-3 has the best antibacterial activity against Staphylococcus aureus and Escherichia coli, Pd NDs-3 was selected as a representative to determine the cell death induced by porous Pd nanoflowers by fluorescent live / dead staining.

[0098] The experimental method is in accordance with the instructions of the kit, as follows: Take 1 mL of the antibacterial test reaction solution (5×10 6CFU / mL), centrifuge at 6000rpm (5000×g) for 5 min, remove the supernatant, add 1mL of sterilized 0.85% NaCl solution to resuspend the bacteria. Repeat this step to wash the bacteria, resuspend the bacteria with 0.5mL of 0.85% NaCl solution, add 5 μL of staining working solution (freshly prepared: take 1μL of NucGreen and 2μL of Eth-Ⅲ, mix well by pipetting, and add 8 μL of 0.85% NaCl solution), incubate at room temperature and avoid light for 15 min, take 10 μL of bacterial suspension and drop it on the slide and cover it with an 18 mm square coverslip to observe the bacteria (NucGreen: Ex= 503 nm / Em= 530 nm; Eth-Ⅲ: Ex= 530 nm / Em= 620 nm), the results are shown in the table below. Fig.17 , 18 .

[0099] like Fig.17 As shown, obvious red fluorescence was observed in Staphylococcus aureus after treatment with Pd NDs-3, indicating that PdNSs-3 has excellent antibacterial activity in inducing the death of Staphylococcus aureus.

[0100] like Fig.18 As shown, obvious red fluorescence was observed in E. coli after treatment with Pd NDs-3, indicating that Pd NSs-3 had excellent antibacterial activity in inducing E. coli cell death.

[0101] 3. Antibacterial mechanism of porous palladium nanoflowers 3.1 Adhesion of porous palladium nanoflowers on bacterial surfaces The experimental method is as follows: take three generations of fresh logarithmic growth phase bacteria (OD600≈0.65), centrifuge at 12000 rpm to remove the supernatant culture medium, then wash and centrifuge with the prepared phosphate buffer, then add PB buffer (pH=4) to disperse and dilute to a bacterial concentration of 5×10 6 CFU / mL, palladium nanomaterials (50 μg / mL) were added, incubated at 37°C for 1 h and then centrifuged. The cells were fixed with 4% paraformaldehyde in a 4°C refrigerator in the dark for 2 h. After being taken out, the cells were shrunk in gradients using ethanol solutions of different concentrations (25%, 50%, 75%, 90%, 100%) and centrifuged again using 100% ethanol (12000 rpm, 2 min). After that, 10 μL was taken and placed on a silicon wafer. After drying overnight, the bacteria were observed using a scanning electron microscope. The results are shown in Fig. Fig.19 .

[0102] like Fig.19As shown in the figure, after treatment with PB buffer (pH = 4), Staphylococcus aureus and Escherichia coli still maintained their original rod-shaped and spherical morphologies, with smooth surfaces and intact cell walls, indicating that PB buffer (pH = 4) is less toxic to these two bacteria. However, when Staphylococcus aureus and Escherichia coli were treated with porous palladium nanoflowers, a large amount of substances could be observed to aggregate and adhere to the bacterial surface, and there was no significant difference in the adhesion ability of porous palladium nanoflowers to Gram-negative bacteria Escherichia coli or Gram-positive bacteria Staphylococcus aureus. It is speculated that this may be due to the fact that the attachment of Pd NDs to the bacterial surface is due to the physical topological interaction of its surface, rather than a chemical mechanism. In addition, it can be observed that the bacterial membrane is destroyed, with irregular and wrinkled morphology, especially for bacteria treated with Pd NDs-3, the damage is more serious.

[0103] like Fig. 20 As shown in the figure, after being treated with PB buffer (pH = 4), Pichia pastoris maintained its original spherical shape, smooth surface, and intact cell wall, indicating that PB buffer (pH = 4) is less toxic to this fungus. However, when Pichia pastoris was treated with Pd NDs-3, a large amount of material was observed to aggregate and adhere to the bacterial surface, the bacterial film was destroyed, the shape was irregular and wrinkled, and the damage was serious.

[0104] 3.2 Porous palladium nanoflowers produce ROS after interacting with bacteria When bacteria were treated with Pd NDs-3, the ROS-sensitive probe DCFH-DA was used to measure the intracellular ROS level. DCFH-DA is a non-fluorescent cell-penetrating probe, but when it is taken up by cells, it is deacetylated by cellular esterases to form 2',7'-dichlorodihydrofluorescein (DCFH), which can interact with ROS to produce fluorescent 2',7'-dichlorofluorescein (DCF). Pd NDs-3 was used as a representative to observe the generation of ROS after the porous palladium nanodendrites interacted with bacteria.

[0105] Experimental method: Take three generations of fresh logarithmic growth phase bacteria (OD600≈0.65), centrifuge at 12000 rpm to remove the supernatant culture medium, then wash and centrifuge with the prepared phosphate buffer, then add PB buffer (pH=4) to disperse and dilute to a bacterial concentration of 5×10 6 CFU / mL, palladium nanomaterials (50 μg / mL) were added, and 2',7'-dichlorodihydrofluorescein diacetate was added to a final concentration of 10 μM after incubation at 37°C for 1 h. After the reaction was protected from light for 30 min, 10 μL of the bacterial suspension was dropped on a glass slide and covered with an 18 mm square coverslip to observe the bacteria (Ex = 488 nm / Em = 525 nm).

[0106] like Fig.21As shown, compared with the Pd-free NDs-3 group, the Pd NDs-3 treated group showed obvious green fluorescence, indicating the formation of ROS in the bacteria.

[0107] In summary, the present invention prepares a porous palladium nanomaterial flower, whose porous structure and rough surface not only have good adhesion ability to bacteria and can cause physical damage to bacteria, but also can effectively shorten the interaction distance between ROS and bacteria, improve the efficiency of oxidase-like enzymes, thereby catalyzing oxygen to produce a large amount of ROS in a short time, which can greatly reduce the amount of nanomaterials used as antibacterial agents. The present invention provides a new strategy for the design of high-efficiency antibacterial nanocomposite materials.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing porous palladium nanoflowers, characterized in that: After the carbon quantum dots and the palladium source are uniformly mixed in water, a reducing agent is added to react to obtain the porous palladium nanoflowers.

2. The preparation method according to claim 1, characterized in that: The particle size of the carbon quantum dots is less than 5 nm.

3. The preparation method according to claim 1, characterized in that: The palladium source includes one or more of palladium chloride, potassium chloropalladate, and chloropalladic acid.

4. The preparation method according to claim 1, characterized in that: The reducing agent is ascorbic acid.

5. The preparation method according to claim 1, characterized in that In the mixed reaction solution, the molar concentration of palladium atoms is 0.1~5 mmol / mL, and the mass concentration of carbon quantum dots is 10~2000 μg / mL; the molar ratio of the reducing agent to the palladium atoms is 6:

1.

6. The preparation method according to claim 1, characterized in that: The reaction temperature is 30-40°C and the reaction time is 0.5-4h.

7. The preparation method according to claim 1, characterized in that: The obtained porous palladium nanoflowers are formed by the aggregation of palladium nanocrystals with a particle size of 6.2nm~7.4nm, and the particle size is above 35.8nm.

8. A porous palladium nanoflower prepared by the method according to any one of claims 1 to 7.

9. Use of the porous palladium nanoflower as claimed in claim 8 as an oxidase-like enzyme.

10. Use of the porous palladium nanoflower as claimed in claim 8 as an antibacterial agent.

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