Ag@Co nanomaterials: Preparation and application in the purification of Yellow River water


 Background

In recent years, nanomaterials have attracted much more attention because of their potential broad-spectrum antimicrobial activity. However, finding biofunctional nanomaterials is still challenging. Therefore,we prepared a new type of recyclable Ag@Co nanomaterials with a very simple method named displacement reaction.
Methods

Ag@Co nanomaterials were prepared in AgNO3 solution with cobalt nanomaterials as the core, while the surface was coated with silver nanomaterials. Ag@Co nanomaterials were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and oscillating sample magnetometer (VSM).In order to evaluate the bacteriostatic effect of Ag@Co nanomaterials, we used qualitative and quantitative methods to assess the bacteriostatic effect of these materials on standard strains of Pseudomonas aeruginosa (ATCC27853, Gram - negative bacterium), Staphylococcus aureus (ATCC 25923, Gram-positive bacterium), Escherichia coli (ATCC 25922, Gram-negative bacterium) and Candida albicans (ATTC 90029, yeast) in Yellow River water in vitro.
Results

We found that the diameter of the Ag@Co nanomaterials was about 100nm, which was exhibited in form of elemental elements and obvious magnetic properties.The results show that Ag@Co nanomaterials have excellent antibacterial effects on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans and the bacteria in the water of the Yellow River.
Conclusions

Ag@Co nanomaterials have a significant antibacterial effect and can be recycled through an external magnetic field to reduce environmental pollution. Interestingly, the reclaimed Ag@Co nanomaterials can still have antibacterial activity and can be reused. These results indicate that Ag@Co nanomaterials may have potential application as disinfectants for water.


Abstract Background
In recent years, nanomaterials have attracted much more attention because of their potential broadspectrum antimicrobial activity. However, nding biofunctional nanomaterials is still challenging. Therefore,we prepared a new type of recyclable Ag@Co nanomaterials with a very simple method named displacement reaction.

Methods
Ag@Co nanomaterials were prepared in AgNO 3 solution with cobalt nanomaterials as the core, while the surface was coated with silver nanomaterials. Ag@Co nanomaterials were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and oscillating sample magnetometer (VSM).In order to evaluate the bacteriostatic effect of Ag@Co nanomaterials, we used qualitative and quantitative methods to assess the bacteriostatic effect of these materials on standard strains of Pseudomonas aeruginosa (ATCC27853, Gram -negative bacterium), Staphylococcus aureus (ATCC 25923, Gram-positive bacterium), Escherichia coli (ATCC 25922, Gramnegative bacterium) and Candida albicans (ATTC 90029, yeast) in Yellow River water in vitro.

Results
We found that the diameter of the Ag@Co nanomaterials was about 100nm, which was exhibited in form of elemental elements and obvious magnetic properties.The results show that Ag@Co nanomaterials have excellent antibacterial effects on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans and the bacteria in the water of the Yellow River.

Conclusions
Ag@Co nanomaterials have a signi cant antibacterial effect and can be recycled through an external magnetic eld to reduce environmental pollution. Interestingly, the reclaimed Ag@Co nanomaterials can still have antibacterial activity and can be reused. These results indicate that Ag@Co nanomaterials may have potential application as disinfectants for water.

Background
The infection and spread of pathogenic bacteria have always been a threat to public health. Although the threat of pathogenic bacteria has been controlled in developed countries, drug-resistant bacteria, new pathogenic bacteria, and genetically mutated superbugs have emerged with the abuse of antibiotics [1] . In recent years, nanoscaled antibacterial materials are expected to become signi cant antimicrobial elements due to their novel physical and chemical properties. Currently, nanoscaled antibacterial materials include TiO 2 , ZnO, CuO, Ca(HO) 2 , MgO, chitosan, silver, and others [2][3][4][5][6][7][8] . Among them, nanocrystalline silver and its compounds can e ciently inhibit or even kill pathogenic bacteria, viruses and other microorganisms, which has the advantage of lower toxicity and side effects and plays an important role in local bacteriostatic therapy. Clinically, nanocrystalline silver-loaded medical products can kill bacteria on wounds to minimize infections, thus promoting the healing of chronic wounds [9] .
Surgical sutures coated with silver-loaded medical products have a stronger ability to inhibit bacterial adhesion compared with uncoated surgical sutures [8] . In addition, nanocrystalline silver-loaded antibacterial materials are also applied in ureteral catheters, ber fabrics, antibacterial plastics, and other materials [10][11][12] . Nanocrystalline silver as the antimicrobial agent is marked by broad-spectrum antibacterial activity, which can kill many kinds of pathogenic microorganisms, and will not lead to microbial resistance [13] .
In terms of sewage treatment, TiO 2 has been used in wastewater treatment systems for a long time; however, excessive accumulation of silver and silver-containing compounds in the body will bring about serious silver poisoning thereby extra time and cost must be paid to deal with these residual chemical materials. Therefore, it is limited in its application prospect with few studies existing on the application of silver-containing compounds in sewage treatment. To solve this problem, people propose that the nanocrystalline silver can be coated on the surface of magnetic materials, and then the silver can be recycled by external magnetic force, easily and e ciently after wastewater treatment. In this course, people can not only e ciently treat wastewater, but also can reclaim the silver-containing compounds to avoid environmental pollution and save costs. Cobalt is a kind of amphoteric metal which is identi ed with good magnetism, similar to iron and nickel in hardness, has suitable tensile strength, machinability, thermodynamic properties and electrochemical behavior. Cobalt especially has obvious advantages in magnetism, being one of the few metals that can remain magnetic after being magnetized once [14] .
In this study, cobalt nanomaterials were used as carriers to prepare silver-loaded Ag@Co nanomaterials in aqueous solution, as shown in Fig. 1-(a). These materials have both the antibacterial bioactivities of silver and the magnetic properties of cobalt to ensure that the nanomaterials can be recycled through the external magnetic eld. Furthermore, the Ag@Co nanomaterials bring out signi cant antibacterial effects on the standard strain, with the growth of bacteria in water of the Yellow River also being inhibited Fig. 1(b),(c). The results show that the magnetic Ag@Co nanomaterials have a signi cant antibacterial effect, and promisingly can be used as a new type of recyclable antibacterial agent.

Treatment of the Yellow River water
Firstly, large debris were removed from the Yellow River water through 200-mesh sieve. Then, the Yellow River water stood for precipitation to obtain the supernatant under sterile conditions. A volume of 100μl Yellow River water supernatant was absorbed and evenly applied on LB solid medium, which was removed to be cultured at 37℃ for 12h. The turbidity of Yellow River water as 6 ~ 7×10 3 CFU/MLand the bacterial assemblage of the Lanzhou section was mainly composed of fecal coliform [15][16] .

Preparation of Ag@Co nanomaterials
70.8mg cobalt nanomaterials and 272mg AgNO 3 powder were accurately weighed and each dissolved in 50mL deionized water. The two solutions were added to a three-pot ask, and the volume of reaction system was increased to 200mL.The mixture was stirred (800rmp) for 4h at room temperature. Ag@Co nanomaterials can be obtained after centrifugation at 5000rmp. The samples were successively washed with deionized water and anhydrous ethanol by ultrasonic and centrifugation for 3 times, and then freezedried for further use.

Characterization of Ag@Co nanomaterials
The morphologies of the synthesized Ag@Co nanoparticle samples were characterized by transmission electron microscope (TEM, Kevex JSM-6701F, Japan). The type of elements, valence and mass ratio of Ag@Co nanomaterials were determined by X-ray photoelectron spectroscopy (XPS, ESCALAB 250, ThermoFisher Scienti c Technology, USA). The phase composition of the Ag@Co nanomaterials was determined by X-ray diffraction (XRD, Bruker D8 Advance). The magnetic properties of Ag@Co nanomaterials were detected by an oscillating sample magnetometer at room temperature (VSM, Lakeshore Cryotronics Inc., Ohio, USA).

Measurements of antibacterial properties of Ag@Co nanomaterials in vitro
The standard strains of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans were selected as representative bacteria for investigating the antibacterial properties of Ag@Co nanomaterials. The minimum inhibitory concentration (MIC) values of Ag@Co nanomaterials were determined by the disc agar diffusion method and broth dilution method. 10mg/ml of Ag@Co nanoparticle suspension were mixed into blank drug-sensitive paper, which was shook overnight to prepare drug-sensitive paper containing Ag@Co nanomaterials. The prepared drug-sensitive papers were pasted on the M-H agar medium inoculated with the above four kinds of standard bacteria overnight to measure the antibacterial effect.
Under sterile conditions, a single colony of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans were selected and diluted with LB liquid medium to reach a level of 1.0×10 5 CFU/ML. 2mL diluted bacterial solution was added to each well of the 24-well plate sequentially. Then, a certain amount of bacteriostatic solution containing Ag@Co nanomaterials was added in order and the concentrations of the bacteriostatic solution was as follows: 0 (positive control, bacteria solution added without Ag@Co nanomaterials), 100, 200, 300, 400, 500, 600, 700, 900, 1000, 2000g/ml, with the last well as a negative control (2ml Ag@Co nanomaterials bacteriostatic solution added); all the groups were with the duplicate holes set. The plates were incubated at 37℃ on a rotary shaker (180 rpm) for 12h. The antibacterial effect of Ag@Co nanomaterials was measured on the next day, and the minimum concentration without bacterial growth was the MIC.
The minimum inhibitory concentration (MIC) of Ag@Co nanomaterials for the Yellow River water: under sterile conditions, the Yellow River water was diluted into the LB liquid medium at 1:100. After that, 2ml diluted Yellow River water LB liquid medium was added to the 24-well plate, and then a certain amount of bacteriostatic solution containing Ag@Co nanomaterials was added sequentially. The bacteriostatic concentration of each well was as follows: 0 (positive control, only bacteria solution added without Ag@Co nanomaterials), 100, 200, 300, 400, 500, 600, 700, 900, 1000, 2000g/ml, with the last well as negative control (2ml Ag@Co nanomaterials bacteriostatic solution added); all the groups were with the duplicate holes set. The plates were incubated at 37℃ on a rotary shaker (180rpm) for 12 h. The antibacterial effect of Ag@Co nanomaterials was measured on the next day, and the minimum concentration without bacterial growth was the MIC.

Measurement of antibacterial properties of Ag@Co nanomaterials reclaimed
Measurement of minimum inhibitory concentration (MIC) of Ag@Co nanomaterials reclaimed for Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans: After the MIC of Ag@Co nanomaterials were measured, the magnets were put under the 24-well plates for 30 min to make sure that Ag@Co nanomaterials were attracted at the bottom of the plate. After slowly discarding bacteria liquid in the 24-well plates, 2ml of diluted bacteria liquid was added. The plates were incubated at 37℃ on a rotary shaker (180rpm) for 12h. The bacteriostatic effect of Ag@Co nanomaterials was observed by naked eyes on the third day, and the minimum concentration without bacteria growth was MIC.
Measurement of minimum inhibitory concentration (MIC) of Ag@Co nanomaterials reclaimed for Yellow River water: After the MIC of Ag@Co nanomaterials for Yellow River water were measured, the magnets were put under the 24-well plates for 30 min to ensure that Ag@Co nanomaterials were attracted to the bottom of the plate. After slowly discarding the bacteria liquid in the 24-well plates, 2 ml diluted bacteria liquid were added. The plates were incubated at 37℃ on a rotary shaker (180 rpm) for 12h. The bacteriostatic effect of Ag@Co nanomaterials was observed by naked eyes on the third day, and the minimum concentration without bacteria growth was MIC.
2.8. Measurements of the reclaim rate of reclaimed Ag@Co nanomaterials 5mg of Ag@Co nanomaterials were precisely weighed and dissolved into a centrifuge tube containing 25mL Escherichia coli dilution solution with a bacterial concentration of 1.0×10 5 CFU/ML. The bacteria were incubated at 37℃ on a rotary shaker (180rpm) for 12h. The next day, the magnet was placed under the centrifuge tube for about 30min to make sure that Ag@Co nanomaterials were attracted to the bottom of the tube. Then slowly discarding the bacteria liquid in the centrifuge tube and dried the left Ag@Co nanomaterials at 60℃ until the centrifuge tube was at constant weight. The reclaimed Ag@Co nanomaterials were weighed, and the reclaim rate was calculated according to the following formula: recovery (%) = recovery amount/input amount*100%.

Characterization of Ag@Co nanomaterials
The morphology of Ag@Co nanomaterials characterized by TEM is shown in Fig. 2-(a). The outer surface of cobalt nanomaterials is covered with a layer of silver element in a "dendritic-like" formation. As shown in Fig. 2-(b), the composition of "dendritic" Ag@Co nanomaterials was relatively uniform in size, with an average particle size about 100nm.
The patterns of the X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) of Ag@Co nanomaterials are shown in Fig. 3. Data displayed in igure 3-(a) suggests that Ag@Co nanomaterials contain the characteristic peaks of both silver and cobalt elements, indicating that that the silver and cobalt elements contained in Ag@Co nanomaterials are successfully combined together. Figure 3-(b) is the full spectrum of XPS, depending on analysis of the element distribution, the surface of Ag@Co nanomaterials contains three elements: O, Ag, and Co, among which Co and Ag accounts for 9.04% and 10.03%, respectively. The data was obtained by calculating the area under the Co and Ag wave peaks. The X-ray photoelectron spectrometer detected the super cial zone of the materials, and the surface of nanomaterials can be oxidized in the air, so O always resided in the Ag@Co nanomaterials [17][18] . Considering that, the patterns of XRD in Ag@Co nanomaterials indicate that only Ag and Co are the components of Ag@Co nanomaterials. The valence distribution of Ag contained in Ag@Co nanomaterials is shown in Fig. 3-(c). The valence of Ag exhibits 3d 5/2 and 3d 3/2 , and its binding energy is 368.4eV and 374.4eV, respectively. These levels of binding energy are consistent with their respective standard electron binding energies. The other positions whose binding energy are 374.43 eV and 368.43 eV correspond to the peak positions of Ag + in Ag 2 O [19] . The valence distribution of CO contained in Ag@Co nanomaterials is shown in Fig. 3-(d). Binding energies of 780.88eV and 796.9eV correspond to the 2p 3/2 and 2p 1/2 valence of Co in Co 3 O 4, respectively, while binding energies of 786.58eV and 803.79eV are the satellite of Co in Co 3 O 4 , and binding energies 778.39eV and 793.49eV correspond to the peaks of the Co 0 valence in elemental cobalt [19] .
The magnetic properties of Ag@Co nanomaterials are crucial for subsequent applications. Placing a magnet next to the container containing the Ag@Co nanomaterials suspension, result in the nanomaterials moving quickly to the side and close to the magnet under the magnetic force. This means that the Ag@Co nanomaterials have good magnetic responsiveness as shown in Fig. 4-(a). At room temperature, VSM was used to detect the magnetic properties of Ag@Co nanomaterials. The magnetic eld range is -30 ~ 30kOe. We found that Ag@Co nanomaterials have magnetic responsiveness under an external magnetic eld, and the magnetization saturation is 70.1emu/g in Fig. 4-(c). No hysteresis was observed when the magnetic moment of the external magnetic eld is 0, which shows that the synthesized Ag@Co nanomaterials are superparamagnetic so that they can be separated from the solution by the external magnetic eld. As shown in Fig. 4-(b), Ag@Co nanomaterials have lower saturation magnetization than cobalt nanomaterials, which may be attributed to the fact that a layer of silver element attached to the surface of cobalt nanomaterials increases the thickness of the shell layer.

River water
The antibacterial effect of Ag@Co nanomaterials on common bacteria in daily life was evaluated through qualitative and quantitative methods. The results are shown in Fig. 5 and Table 1: Ag@Co nanomaterials have good antibacterial effects on Staphylococcus aureus 3-(a), Escherichia coli 3-(b), Pseudomonas aeruginosa 3-(c) and Candida albicans 3-(d); Ag@Co nanomaterials have signi cantly better antibacterial effects on Pseudomonas aeruginosa, Candida albicans and Staphylococcus aureus than Escherichia coli, and the diameter of the inhibition zone is 12.8 ± 0.4, 15.2 ± 0.4, 12.6 ± 0.5, 10.2 ± 0.4mm, respectively.
From Table 1, we found that Ag@Co nanomaterials can achieve excellent antibacterial effects on general standard strains, and that the reclaimed Ag@Co nanomaterials could be reused still have good antibacterial effects on these bacteria. As well, Ag@Co nanomaterials can achieve excellent antibacterial effects on the Yellow River water, and the reclaimed Ag@Co nanomaterials still have antibacterial effects when reused.

Discussion
Nanobiotechnology is an emerging eld; especially in the eld of medicine. A large number of medicines get revived and applied widely due to the further study of nanomaterials, which have made great contributions to human life and health. Bene tting from the emergence of nanotechnology, common antibacterial materials are remoulded to be nanoscale antimicrobial materials which can be combined with antimicrobial carriers through certain methods and technologies, to be effective. Metal nanoantibacterial agents include many other heavy metal ions such as Ag + , Zn 2+ , Cu 2+ , Hg 2+ and many other heavy metal ions, among which Ag + is the antibacterial agent showing the best antibacterial effect and the least toxicity to the human body. Hence the most widely studied antibacterial agents are those containing silver ions. Recent studies have shown that silver ions have a strong killing effect on 12 kinds of gram-negative bacteria, 8 kinds of gram-positive bacteria and 6 kinds of molds. Due to its excellent antibacterial properties, silver has the potential to be an antibacterial material [20] . Magnetic nanomaterials have the advantages of uniform particle size, large surface area, superparamagnetism, and are widely used in cemented carbide, sewage treatment, batteries, permanent magnet materials, diamond tool manufacturing and other industries, as well as in the elds of catalysts, magnetic materials, absorbing materials, ceramics, and more [21][22] . Among them, compared with iron and nickel nanomaterials, cobalt nanomaterials have stronger magnetic properties and are easier to agglomerate and oxidize.
In this study, cobalt nanomaterials as the core Ag@Co nanomaterials can reduce silver ions in a AgNO 3 solution to become elemental silver which can be coated on the surface of cobalt nanomaterials. The prepared Ag@Co nanomaterials have a uniform particle size of about 100nm, form as "dendritic" so that increasing the materials' surface area easily have contact with the outside world. In addition, the presence of silver on the surface of Ag@Co nanomaterials is higher, which ensures that the materials have excellent antibacterial effects on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans and the bacteria in the water of the Yellow River. Moreover, Ag@Co nanomaterials have strong magnetic properties because of the existence of cobalt. Over the course of sewage treatment, Ag@Co nanomaterials can not only inhibit the growth of bacteria, but also can be reclaimed by the external magnetic eld and the reclaim rate is up to 98%, hence reducing the toxicity of metal silver. Surprisingly, the reclaimed Ag@Co nanomaterials still have a good antibacterial effect. In the long run, the reclaimed Ag@Co nanomaterials technology will greatly save enterprise production costs, and do not have side effects on the surroundings to reduce environmental pollution and disposal costs. The Ag@Co nanomaterials do signi cantly increase economic bene ts, save energy and create bene ts for society such that the materials can be potential candidates for sewage treatment.

Conclusion
In conclusion, Ag@Co nanomaterials have both the antibacterial bioactivities of silver and the magnetic properties of cobalt to ensure that the nanomaterials can be recycled through the external magnetic eld. Furthermore, the Ag@Co nanomaterials bring out signi cant antibacterial effects on the standard strain, with the growth of bacteria in water of the Yellow River also being inhibited. These also makes it possible to serve as a new type of recyclable antibacterial agent.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Con icts of interest
The authors declare no con ict of interest.
Authors' contributions GZ conceived this project, designed the experiment and reviewed the manuscript. XS designed the experiment. YZ designed and performed the experiment, analyzed and interpreted the data and drafted the manuscript. YL, SH, QG and YZ, LK, XZ participated in performing the experiment. All authors read and approved the fnal manuscript.