CN115812895A - Application of biological nano zero-valent palladium material with high photothermal effect in preparation of heating product - Google Patents
Application of biological nano zero-valent palladium material with high photothermal effect in preparation of heating product Download PDFInfo
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Abstract
The invention discloses application of a biological nano zero-valent palladium material with high photothermal effect in preparation of a heating product. The bacillus megaterium Y-4 is utilized to synthesize the biological nano palladium with high photo-thermal effect, and the biological nano palladium is used for antibiosis, food heating and the like. At power densities of 1.35, 2.56 and 3.14W/cm 2 Under the near infrared irradiation, the final temperature of 20mg/L ultrasonic biological Pd-NPs reaches 55 ℃, 78 ℃ and 90 ℃, and the heating performance is good.
Description
The technical field is as follows:
the invention belongs to the technical field of microbial nano materials, and particularly relates to biological nano palladium with high photothermal effect synthesized by bacillus megaterium Y-4, which is used for antibiosis, food heating and the like.
Background art:
it is well known that the most common and effective method for preventing and treating bacterial infections is antibiotic therapy. However, the continued emergence and rapid spread of antibiotic strains has become one of the most serious threats to human health worldwide due to long-term abuse of antibiotics. There is an urgent need to develop a new strategy to address antibiotic-resistant bacteria. Heating processes, such as pasteurization, have been used as a physical antimicrobial means, i.e., the bacteria will be sterilized at temperatures above 55 ℃ due to denaturation of the heat shock proteins until penicillin is discovered. Therefore, development of novel nanomaterials having high biocompatibility is pursued. Photothermal therapy (PTT) involves the conversion of Near Infrared (NIR) light into heat using a photothermal transducer to kill bacteria. It requires shorter treatment times than traditional antibiotic treatments and can avoid the appearance of resistance. Thus, PTT is considered a safe and effective strategy for treating bacterial infections.
For this reason, due to the specific size, shape, composition, larger surface area to volume ratio and purity of the individual components of nanoparticles, efforts have been made in recent years to develop novel nanomaterials, in particular gold nanomaterials, with good photothermal effects. Different gold nanoparticles, including nanorods, nanoshells, nanocages, and nanostars, have been synthesized with excellent ability to convert light energy to thermal energy in the NIR region. However, some of them have disadvantages of poor photo-thermal stability and complicated synthesis process.
Palladium nanostructures have remarkable chemical and optical properties and recently become new candidates for photothermal sensors. Compared with Au nanomaterials, pd nanostructures exhibit excellent photo-thermal stability and high optical extinction coefficient. Studies have shown that the photothermal conversion efficiency of Pd-NPs depends on size and morphology. To achieve high photothermal conversion efficiency, different palladium nanostructures, such as porous palladium nanoparticles and palladium nanosheets, have been synthesized. In addition, the Pd-NPs are modified by surface ligands to improve the water stability of the Pd-NPs so as to improve the photothermal effect. Although these strategies achieve the goal of improving the performance of Pd-NPs in certain respects, there are a number of disadvantages, such as the use of toxic solvents, the production of harmful by-products, and/or the need for complex multiple synthetic steps. Therefore, there is an urgent need to find a new method for synthesizing environmentally friendly and biocompatible Pd-NPs having good photothermal efficacy.
Microbial biosynthesis of nanoparticles is considered a clean, non-toxic and environmentally acceptable green chemical process. It has been reported that many microorganisms are capable of reducing Pd (II) to Pd (0) without the addition of toxic reducing agents and further promote the nucleation and growth of Pd-NPs. We have found that Bacillus megaterium Y-4, a bacterium that grows rapidly under aerobic conditions, is effective in the bioreduction of Pd (II) to Pd (0). Recently, these biosynthetic Pd-NPs have been extensively explored in catalyzing organic synthesis and degradation of environmental pollutants. However, the photothermal conversion properties and the biological application potential of the microbial Pd-NPs are rarely explored.
In this study, we successfully used Bacillus megaterium Y-4 to synthesize the microorganism Pd-NPs and systematically explore its unique physicochemical properties. It has been demonstrated that ultrasonic treatment of the bioreduced Pd-NPs gives materials with higher absorption in the NIR region and better photothermal conversion efficiency. The prepared material shows excellent biocompatibility and antibacterial activity under near infrared radiation. In less than 10 minutes, the sterilization efficiency of the low-dose Pd-NPs (20 mg/L) on staphylococcus aureus and escherichia coli is 99.99%. These results indicate that synthetic Pd-NPs have great potential as PTT reagents for rapid disinfection applications.
The invention content is as follows:
the first purpose of the invention is to provide the application of the biological nano zero-valent palladium material with high photothermal effect in the preparation of heating products, the biological nano zero-valent palladium material has the disinfection efficiency of low dose Pd-NPs (20 mg/L) on staphylococcus aureus and escherichia coli of 99.99 percent in less than 10 minutes, the reaction condition is mild, the required cost is low, and the biological nano zero-valent palladium material is environment-friendly.
Therefore, the invention provides the application of the biological nano zero-valent palladium material with high photothermal effect in preparing a heating product.
Preferably, the heating product is used for heating, heating food or antibacterial products, etc.
Preferably, the biological nano zero-valent palladium material with high photothermal effect is obtained by reducing Pd (II) with Bacillus megaterium Y-4, and then obtaining the biological nano zero-valent palladium material with high photothermal effect by ultrasonic.
Further preferably, the biological nano zero-valent palladium material with high photothermal effect is 148mg/L B.megaterium Y-4, 100mg/L Pd (II) is reduced under 5mM sodium formate and anaerobic condition, biological nano zero-valent palladium is obtained after reaction for 12h at 30 ℃, and then the biological nano zero-valent palladium material with high photothermal effect is obtained after ultrasonic treatment for 20 min.
Preferably, the application of the biological nano zero-valent palladium material with high photothermal effect combined with near infrared irradiation in heating, food heating or antibacterial products is provided.
Further preferably, the biological nanometer zero-valent palladium material with high photothermal effect is irradiated in near infrared for heating or near infrared irradiation of a mixture such as a solution containing the biological nanometer zero-valent palladium material with high photothermal effect and bacteria to sterilize.
Preferably, 808nm near infrared light is used, and the power density is 1.35-3.14W/cm 2 Irradiating 20-40 mg/L solution of biological nano zero-valent palladium material and bacteria with high photothermal effect for over 10 min.
Preferably, the antimicrobial product is a product against escherichia coli or staphylococcus aureus.
Compared with the prior art, the invention has the following beneficial effects:
(1) At power densities of 1.35, 2.56 and 3.14W/cm 2 Under the near infrared irradiation, the final temperature of 20mg/L ultrasonic biological Pd-NPs respectively reaches 55 ℃, 78 ℃ and 90 ℃, and the heating performance is good;
(2) The ultrasonic biological Pd-NPs in the concentration range of 10-80 mg/mL can keep the cell activity of >90% of human embryonic kidney (HEK 293), and the synthetic biological Pd-NPs have good biocompatibility;
(3) The invention is 1.35W/cm 2 Under the near-infrared radiation power, when the time is more than 10min and the concentration is 20mg/L biological Pd-NPs, the bacteriostasis rates of the bacillus coli and the staphylococcus aureus can respectively reach 99.99 percent and 99.9998 percent.
Drawings
FIG. 1 is a representation of the Bacillus megaterium Y-4 synthetic organisms Pd-NPs: TEM images of biological Pd-NPs without sonication (A and C) and sonication (B and D); XPS spectra of biological Pd-NPs without sonication (E) and sonication (F).
FIG. 2 is photo-thermal properties of biological Pd-NPs, (A) UV-vis-NIR absorption spectra of biological Pd-NPs; (B) Temperature of sonicated biological Pd-NPs suspension (20 mg/L) at different NIR radiation power as a function of time; (C) In NIR radiation (1.35W/cm) 2 ) The temperature of biological Pd-NPs with different concentrations changes along with time; (D) Three switching cycles (1.35W/cm) 2 ) Photothermal cycling curves of medium Pd-NPs (20 mg/L).
FIG. 3 is a biocompatibility assessment of biological Pd-NPs
FIG. 4 is a graph of antimicrobial activity of sonicated biological Pd-NPs, (A) E.coli and (B) Staphylococcus aureus relative antimicrobial efficiencies under different treatments; (C) Plate counts of E.coli and S.aureus after different treatments; a first group: coli, no irradiation; second group: escherichia coli with near-infrared radiation power of 1.35W/cm 2 (ii) a Third group: staphylococcus aureus, no radiation; and a fourth group: staphylococcus aureus, near infrared radiation power 1.35W/cm 2 。
FIG. 5 is an image of overlapping fluorescence (green: all cells; red: dead cells) of E.coli and S.aureus stained with SYTO 9/PI after different treatments.
FIG. 6 shows the irradiation power of the ultrasonic-treated Pd-NPs at 1.35W/cm 2 Antibacterial activity of (A) Escherichia coli and (B) Staphylococcus aureus after different treatment times; (C) Coli and (D) Staphylococcus aureus with sonicated Pd NPs (20 m)g/L) at an irradiation power of 1.35W/cm 2 Next treatment for relative viability over several cycles.
Detailed Description
The following is a further description of the present invention in conjunction with embodiments thereof, and is not intended to limit the present invention.
Example 1 screening and identification of Bacillus megaterium Y-4
Weighing 10.0g of sludge of an electronic waste factory, putting the sludge into a 250mL conical flask, adding 100mL of fresh LB culture medium, adding a certain amount of sodium tetrachloropalladate, sealing the mixture by using an air sealing film, and putting the mixture into a shaking table with the rotating speed of 150r/min and the temperature of 30 ℃ for acclimatization and enrichment culture. The 10mg/L Pd (II) is added in the acclimatization process at the beginning, then the acclimatization process is carried out for three days, the 10mg/L is sequentially increased, each concentration is acclimatized for three days, and the final Pd (II) concentration is 100mg/L, and the acclimatization process is carried out for about 30 days. 1mL of domesticated mixed bacterial liquid is taken according to the proportion of 10 -1 ……10 -7 Diluting, uniformly coating 100 μ L of the diluted solution on a plate, culturing in an incubator at 30 deg.C for 12h, selecting single colony, performing liquid culture, diluting, coating, further separating and purifying until the colony morphology is consistent, and preserving with glycerol. Then, the effect of each bacterium on reducing Pd (II) is examined, the bacterium with good effect on reducing Pd (II) is further purified and cultured in an enlarged way, and then the strain is identified, so that the strain Y-4 is obtained.
The strain Y-4 is aerobic bacteria, gram-positive, rod-shaped, round at the tail end and capable of moving. Pure culture of the strain Y-4, extraction of total DNA of the strain, PCR amplification of 16S rDNA, sending the amplified product to Guangzhou branch company of Biotechnology Limited of Beijing OngOptike for sequence determination, and splicing the positive and negative sequences to obtain a 16S rDNA gene sequence with the length of 1493bp, as shown in SEQ ID NO. 1. The similarity of the strain to Bacillus megaterium strain MN509794.1 is 99.93 percent by BLAST comparison on NCBI website (https:// www.ncbi.nlm.nih.gov /), and the strain is named as Bacillus megaterium Y-4.
The strain is disclosed in The literature of The effect of biological and enzymatic factors on Pd (II) uptake and reduction by Bacillus megaterium Y-4, which is also owned by The applicant and guaranteed to be provided to The public within 20 years from The filing date.
Example 2B. Megaterium Y-4 Synthesis of biological Pd-NPs and characterization thereof
B, carrying out slant culture and activation on megaterium Y-4 for 1-2 days, then taking a ring to a 250mL conical flask filled with 100mL liquid LB culture medium, sealing the conical flask by using an air sealing film, placing the conical flask in a constant-temperature oscillation incubator, culturing at the temperature of 30 ℃ and at the speed of 150r/min, centrifuging the cultured bacterial liquid for 5min at the speed of 4000r/min, removing a supernatant culture medium to obtain thalli, washing by using a sterile phosphate buffer solution (pH = 7.0), and centrifuging and cleaning twice at the speed of 4000r/min to obtain the thalli. Then 148mg/L B.megaterium Y-4 reduces 100mg/L Pd (II) under 5mM sodium formate and anaerobic condition, and reacts for 12h under the condition of 30 ℃ to obtain biological nano zero-valent palladium (Pd-NPs). And carrying out ultrasonic treatment for 20min on the biological nano zero-valent palladium (Pd-NPs) to obtain the ultrasonic-treated biological nano zero-valent palladium (Pd-NPs).
TEM images (FIGS. 1A, 1C) show that the biological Pd-NPs obtained are mainly distributed in the periplasmic space of the cells, and Pd-NP aggregation is observed, B.megaterium Y-4 reduces Pd 2+ The synthetic Pd-NPs inevitably precipitate and aggregate with increasing reaction time, which may have a negative effect on the optical properties thereof. Thus, sonication releases the Pd-NPs from the microbial host. TEM images of Pd-NPs after 20min of sonication are shown in FIG. 1B and FIG. 1D. The separated Pd shows better dispersibility, which may be beneficial to the improvement of the physicochemical property. And evaluating the chemical states of the Pd-NPs before and after ultrasonic treatment by adopting an XPS characterization method. As shown in FIGS. 1E and 1F, the binding energies centered around 339.82eV and 334.57eV are ascribed to Pd 0 Pd of 3d/2 And Pd 5d/2 The binding energies centered at 341.51eV and 336.26eV are assigned to Pd 2+ Pd of 3d/2 And Pd 5d/2 . According to Pd 0 And Pd 2+ Peak area of, pd of sonicated and non-sonicated samples 0 The contents were 76.9% and 75.8%, respectively, indicating that the sonication had no effect on the chemical state of the Pd-NPs.
EXAMPLE 3 photothermal Properties of biological Pd-NPs
The ultrasound at the same concentration prepared in example 2 was measured by an ultraviolet-visible spectrophotometerUltraviolet-visible-near infrared absorption spectra of Pd-NPs and non-ultrasonic Pd-NPs. As shown in fig. 2A, the sonicated Pd-NPs exhibited stronger absorbance compared to the non-sonicated samples, which is probably due to the improved water dispersibility of the Pd-NPs released from b.megaterium Y-4. In order to systematically evaluate the photothermal properties of sonobiological Pd-NPs, 1mL of Pd-NPs of different concentrations were irradiated with lasers of different power densities at 808nm, and the temperature changes were recorded with a digital thermometer. The results showed that the power densities were 0.015, 1.35, 2.56 and 3.14W/cm, respectively 2 The final temperatures of 20mg/L ultrasonic Pd-NPs (FIG. 2B) reached 41, 55, 78 and 90 ℃ respectively. At a power density of 1.35W/cm 2 The final temperatures of 5.5, 11, 20 and 40mg/L ultrasonic Pd-NPs reached 41, 45, 56 and 62 ℃ respectively under near infrared radiation (FIG. 2C). There was no significant change in the temperature of the control PBS solution (figure 2C). As expected, the sonobiological Pd-NPs have good photothermal conversion capability, and the photothermal effect is related to the material concentration and the optical density. The photostability of the ultrasonic Pd-NPs was further investigated at 20min heating and cooling cycles, and the results are shown in FIG. 2D. After 5 cycles, the photothermal properties of the ultrasonic Pd-NPs are almost unchanged compared with those before treatment, and the photothermal properties of the ultrasonic Pd-NPs are slightly reduced. This is probably due to further aggregation of the non-sonicated Pd-NPs under photothermal effect. These results indicate that biological Pd-NPs have good photo-thermal stability and are a promising photo-thermal reagent.
EXAMPLE 4 antimicrobial Activity of sonicated Pd-NPs
Before the sonobiological Pd-NPs were used to kill bacteria, their cytotoxicity was first studied. Human embryonic kidney (HEK 293) cells were incubated with various concentrations of the biological Pd-NPs prepared in example 2 for 24 hours, and then cell viability was assessed using the CCK-8 method (FIG. 3). Pd-NPs in the concentration range of 10-80 mg/mL can maintain the cell viability of >90%, and the synthetic biological Pd-NPs have good biocompatibility.
We further investigated the in vitro bactericidal activity of sonicated biological Pd-NPs against e.coli and s.aureus by plate counting (figure 4). Ultrasound was used at different concentrations (0, 5.5, 11 and 20 mg/L)Biological Pd-NPs are irradiated in near infrared (808nm, 1.35w/cm) 2 ) And treating the bacteria in the absence of light. With the increase of the concentration of Pd-NPs, the number of viable colonies of group II (e.coli, near infrared irradiation group) and group iv (s.aureus, near infrared irradiation group) decreased sharply, while the number of viable colonies of group I (e.coli, non-irradiation group) and group III (s.aureus, non-irradiation group) did not change significantly. There was almost no colony growth on agar of groups II and IV treated with 20mg/L Pd-NPs suspension. In contrast, in the control group (Pd-NPs: 0 mg/L), a large number of colonies survived on the agar plate regardless of irradiation with or without irradiation with near-infrared light.
The inactivation of the sonicated biological Pd-NPs on E.coli and S.aureus under near infrared irradiation was further determined using a SYTO 9/Propidium Iodide (PI) double stain kit (dead bacteria: red fluorescence; all fines: green fluorescence). As shown in FIG. 5, in the absence of irradiation, the bacteria incubated with sonicated biological Pd-NPs were mostly stained green, indicating that they were very bacterially active. However, under near infrared irradiation, after 10 minutes, the number of red-stained bacteria was almost equal to the number of green-stained bacteria, indicating that Escherichia coli and Staphylococcus aureus were almost completely inactivated. To reveal that the bacteria were destroyed by the photothermal properties of sonobiological Pd-NPs, a control analysis was further performed by staining cells with only near infrared treatment. Almost all bacteria were stained only with green fluorescence, indicating that the effect of near infrared light on cell viability is negligible. These results strongly support that sonobiological Pd-NPs can kill bacteria through their good photothermal conversion properties. These results indicate that the main factor for inactivating bacteria is not near infrared or Pd-NPs, but rather the excellent photothermal effect of biological Pd-NPs.
At 1.35W/cm 2 Under the near-infrared radiation power, the change of the bacteriostatic activity of the ultrasonic biological Pd-NPs with different concentrations on escherichia coli and staphylococcus aureus along with time is researched (fig. 6A and 6B). Along with the extension of 808nm laser irradiation time, the antibacterial efficiency is gradually improved. When the time is longer than 10min and the concentration is 20mg/L biological Pd-NPs, the bacteriostasis rates of the bacillus coli and the staphylococcus aureus can respectively reach 99.99 percent and 99.9998 percent. The results show that the sonobiological Pd-NPs are applied to Escherichia coli andthe staphylococcus aureus can achieve the rapid sterilization effect within 10min>99.99%). The material has good photo-thermal stability, so that the reusability of the material is researched. As shown in fig. 6C and 6D, even after four cycles, over 94.1% of escherichia coli and 99.5% of staphylococcus aureus were killed, indicating that the ultrasonic Pd-NPs can be repeatedly used for antibacterial applications.
Claims (8)
1. The application of the biological nano zero-valent palladium material with high photothermal effect in preparing heating products.
2. Use according to claim 1, wherein the heated product is for heating, heating food or an antimicrobial product.
3. The application of claim 1 or 2, wherein the biological nano zero-valent palladium material with high photothermal effect is obtained by reducing Pd (II) with Bacillus megaterium (Y-4) to obtain biological nano zero-valent palladium, and performing ultrasonic treatment to obtain the biological nano zero-valent palladium material with high photothermal effect.
4. The application of claim 3, wherein the biological nano zero-valent palladium material with high photothermal effect is 148mg/L B.megaterium Y-4, 100mg/L Pd (II) is reduced under 5mM sodium formate and anaerobic condition, the biological nano zero-valent palladium is obtained after reaction for 12h at 30 ℃, and then the biological nano zero-valent palladium material with high photothermal effect is obtained after ultrasonic treatment for 20 min.
5. Use according to claim 1 or 2, characterized in that it is the use of a bionano zero valent palladium material with high photothermal effect in combination with near infrared irradiation for heating, heating food or antimicrobial products.
6. The use according to claim 5, characterized in that the biological nano zero-valent palladium material with high photothermal effect is irradiated in near infrared for heating or near infrared irradiation of a mixture containing the biological nano zero-valent palladium material with high photothermal effect and bacteria for sterilization.
7. The use of claim 6, wherein the near infrared light of 808nm is used, and the power density is 1.35-3.14W/cm 2 Irradiating 20-40 mg/L solution of biological nano zero-valent palladium material and bacteria with high photothermal effect for over 10 min.
8. Use according to claim 2, 6 or 7, wherein the antimicrobial product is an escherichia coli or staphylococcus aureus resistant product.
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