Dissolvable Trimolybdate Nanowires as Ag Carriers for High-Efficiency Antimicrobial Applications

Elimination of bacteria and other microbes effectively is important to our daily life and a variety of medical applications. Here, we introduce a new kind of trimolybdate nanowires, namely Ag2−x(NH4)xMo3O10 · 3H2O, that carry a large amount of Ag atoms in the lattice and Ag-rich nanoparticles on the surface. These nanowires can eliminate bacteria of E. coli, Staphylococcus aureus, and unknown microbes in raw natural water with high efficiency. For example, they can inactivate more than 98% of E. coli with a nanowire concentration of only 5 ppm in the solution. The excellent sterilization performance is attributed to the combined effects of Ag ions, Mo ions, and Ag-rich nanoparticles of the Ag2−x(NH4)xMo3O10 · 3H2O nanowires. These nanowires are not dissolvable in deionized water but can be dissolved by the metabolic materials released from bacteria, making them attractive for many biological applications.

Here, we introduce a new kind of trimolybdate nanowires, namely Ag 2−x (NH 4 ) x Mo 3 O 10 •3H 2 O, that carry a large amount of Ag atoms in the lattice and Ag-rich nanoparticles on the surface.Similar to previously reported powders of Ag 2 MoO 4 , Ag 2 Mo 2 O 7 , Ag 6 Mo 10 O 33 , Ag 2 Mo 3 O 10 •1.8H 2 O, and so forth [34][35][36][37], the Ag 2−x (NH 4 ) x Mo 3 O 10 •3H 2 O nanowires show strong antimicrobial properties, for example, they kill E. coli, Staphylococcus aureus, and unknown microbes in raw natural water with high efficiency.The nanowires are not dissolvable in deionized water; however they could be dissolved by the metabolic chemicals released from bacteria, making them attractive for many biological applications.

Materials and Methods
The Ag 2−x (NH 4 ) x Mo 3 O 10 •3H 2 O nanowires and other nanomaterials were synthesized from a mixed aqueous solution of (NH 4 ) 6 Mo 7 O 24 •4H 2 O (purity 99.999%) and AgNO 3 (purity 99.999%) that were separately dissolved in deionized water.Their mass ratio in the mixture, ranging from 10 : 1 to 1 : 3, had been systematically studied.The pH value of the starting solutions was controlled with drops of diluted HNO 3 acid.The mixed solution in an open beaker was immersed into a water bath kept at a set temperature between 20 and 80 • C, and a magnetic stirrer was used during the whole reaction process.In minutes yellowish-white precipitates were obtained from the mixed solution, and gradually the whole solution turned into porous, paste-like precipitates with some residual water.After centrifuging and rinsing the precipitates with deionized water for 3-6 cycles, the samples were dried in air and characterized by means of scanning electron microscopy (SEM, Tecnai XL30F), X-ray diffraction (XRD, Rigaku D/max-2400) for morphology, transmission electron microscope (TEM, Tecnai G20, 200 kV) for imaging, in situ energy-dispersive X-ray (EDX) and selected area electron diffraction (SAED), and Cu spectra for surface binding energy of the Ag atoms.Thermogravimetric analysis (TGA, Q600) was performed in the ambient of pure nitrogen gas.
For the cytotoxicity test, 293E cell line was chosen to be the human cells sample.First, the cells sample was cultured with nutrient medium of DMEM, which contained 10% fetal calf serum to reach a concentration of 60-80% in a culture dish with diameter of 10 cm.Next, the nutrient medium was moved away and the cells were washed out with 2 mL PBS.Then trypsogen was added into the sample for 40 seconds.The pseudo pods of the adherent cells retracted and the cells turned into round shapes.Subsequently, 3 mL nutrient medium of DMEM was added into the sample and the solution was blown with a pipette to suspend cells, meanwhile the number of cells was counted with a count plate.After that, about 3 × 10 5 cells were taken to each of 12well plates, and the solution in each well was supplemented to 1 mL with DMEM and stirred until being uniform.At last, the sample was placed at an incubator at 37 • C and 5% CO 2 , adding in nanowires with certain concentrations.The statement of the cell samples was observed closely after different intervals.For the LB plate antibacterial experiments, the E. coli DH5a samples were inverted with PCI-neo-GFP plasmid, and the number of colonies for each trial was counted.The samples were cultured in nutrient Agar (N.A.) at 37 • C for 96 hours.For the skin safety experiments, 4 rabbits weighing 2.3-2.5 kg were used.The temperature and humidity of the rabbit hutch were kept at 18-26 • C and 40-61%, respectively.Hairs on both sides of the spine of each rabbit were totally sheared away, leaving two pieces of clean skin, each about 3 square centimeters.On one piece of the clean skin, 0.5 mL of 1000 ppm Ag-doped nanowires in deionized water was smeared uniformly, while the other piece was left untreated for contrast.

Results and Discussion
The nanowires were synthesized at 30 • C from a mixed aqueous solution of (NH 4 ) 6 Mo 7 O 24 •4H 2 O and AgNO 3 .Various phases of Ag-rich trimolybdate nanomaterials could be obtained in a controlled way when the pH value of the starting solution was adjusted.Figure 1 shows in the left panel 5  XRD and TGA data that will be discussed in the following sections.
Without adding any acid, the mixture solution of (NH 4 ) 6 Mo 7 O 24 •4H 2 O and AgNO 3 naturally maintained its pH value in a narrow range of 4.3-4.7 at 30 • C in the whole reaction process.This was attributed to the existence of NH 4 + ions in the solution [38,39].Synthesized under this condition, the end products were uniform and long nanowires, as shown in Figure 1(e).
The blue line in Figure 2(a) plots the XRD spectrum of the nanowires shown in Figure 1(e).It reveals a single crystalline phase that matches well to the tetragonal phase of (NH 4 ) 2 Mo 3 O 10 (JCPDS 79-1905), peaks of which are marked as black lines in Figure 2, with slight offsets.The lattice constants of (NH 4 ) 2 Mo 3 O 10 are a = 1.3182 nm, b = 0.7589 nm, and c = 0.9286 nm.The lattice constants of our tetragonal nanowire phase are calculated to be a = 1.336 nm, b = 0.754 nm, and c = 0.921 nm, where a becomes 1.35% bigger and b and c become 0.65% and 0.82% smaller, respectively.As a result, most lattice spacings of our nanowires are larger than their counterparts of (NH 4 ) 2 Mo 3 O 10 (see Table 1), while the spacings of ( 212) and (400) are smaller, marked by black triangles in Figure 2(a).Also, the XRD spectrum of our samples matches well to that of NaNH 4 Mo 3 O 10 •H 2 O (JCPDS 36-0335, not shown here), and again with slight offsets, indicating the similarity among these three kinds of tetragonal lattices.This is the first evidence that in our nanowires Ag atoms have taken the lattice positions.
The TGA data of the samples also reveal that Ag atoms substitute the (NH 4 ) + sites in the crystalline lattices.When the Ag-doped trimolybdate nanowires were used in a high concentration, they could efficiently eliminate the growth of bacteria.Figure 3 shows the results of a culture experiment using Lysogeny broth (LB) plate, a more quantitative test for the antibacterial ability of the Ag 2−x (NH 4 ) x Mo 3 O 10 •3H 2 O against E. coli DH5a.It shows that 2 ppm of nanowires in the sample is the critical value of antibacteria.When the concentration increased to 5 ppm, the number of colonies decreased to 2% of that without nanowires.That is, as much as 98% of the bacteria are inhibited.
We have tested the cytotoxicity of the as-synthesized nanowires to find out the concentration range in which the nanowires are safe to human cells and at the same time have an effective antimicrobial effect [40].Here, 293E cell line was chosen to be the human cells sample.The results are shown in Figure 6.The blue (dark and light) regions show the concentration range in which nanowires are friendly to human cells but cannot inhibit the growth of microbes effectively.In the red (dark and light) regions the nanowires are antimicrobial but also do harm human cells.The green region is the concentration window in which the nanowires are both friendly to human cells and have an effective antimicrobial effect.The results show that the safety window of concentration depends much on the reaction time duration.Within 4 hours, the ratio of the highest to the lowest concentrations in the safe region is 100, and this ratio decreases to 15 at 24 hours and 2 at 96 hours.
Morphology imaging and SAED analyses of the assynthesized Ag 2−x (NH 4 ) x Mo 3 O 10 •3H 2 O nanowires with a TEM showed that the nanowires had a single crystalline structure with a growth direction [101] (inset of Figure 4).On the surface of the nanowires, a large amount of randomly distributed dark nanoparticles were observed.The in situ EDX spectra of individual nanowires revealed that, when the electron-beam probe was moved from the center to the edge of the same nanowire, the relative atomic ratio of Ag over Mo was remarkably increased, as typically shown in We note that both nanowires of pure (NH 4 ) 2 Mo 3 O 10 and NaNH 4 Mo 3 O 10 •H 2 O phases are easily dissolvable in deionized water [39], while the nanowires shown in Figures 1(c), 1(d), and 1(e) are not.However, they can be dissolved in mild acidic solutions.As shown in Figure 5, we have observed clear evidence that these nanowires could be dissolved by the chemicals released from the bacteria during their metabolism processes.

Figure 1 :
Figure 1: Left panel, shows typical SEM micrographs of 5 kinds of samples synthesized at 30 • C under varying pH values of 1.0, 2.0, 3.0, 4.0, and 4.3-4.7,respectively.Right panel is their corresponding XRD spectra.The peaks for NH 3 (MoO 3 ) 3 phase are marked by solid red triangles in (b)-(d).Shown in (e), without adding any acid, the mixture solution of (NH 4 ) 6 Mo 7 O 24 •4H 2 O and AgNO 3 naturally maintained its pH value in a narrow range of 4.3-4.7 in the whole reaction process.

Figure 2 :
Figure 2: Experimental XRD data (a) and TGA data (b) of Ag 2−x (NH 4 ) x Mo 3 O 10 •3H 2 O nanowires.The two triangles in (a) indicate the peaks having opposite offset direction to that of the rest peaks, as compared to XRD data of (NH 4 ) 2 Mo 3 O 10 (see the Supplementary figure in the Supplementary Material available online at doi: 10.5402/2012/539601).

Figure 2 (Figure 3 :
Figure 3: Results of LB plate culture experiment of the Ag 2−x (NH 4 ) x Mo 3 O 10 •3H 2 O nanowires against E. coli DH5a.It shows that 2 ppm is the critical concentration for antibacteria.When the concentration increases to 5 ppm, the number of colonies decreases to 2% of that without nanowires.

Figure 4 :
Figure 4: (a) A low magnification TEM image of Ag 2−x (NH 4 ) x Mo 3 O 10 •3H 2 O nanowires.The inset is a typical SAED of a single nanowire, showing single crystalline phase and the growth direction [101] of each nanowire.(b) A close look at a nanowire revealing a large amount of nanoparticles.(c) and (d) EDX spectra of the center region and the edge region of the same nanowire, respectively.

Figure 5 :
Figure 5: Optical images of 3 samples that were spread on the same kind of N.A. base and cultured under the same conditions at 40 • C for 18 hours.G1: pure Staphylococcus aureus bacteria.G2: Staphylococcus aureus bacteria mixed with dry Ag 2−x (NH 4 ) x Mo 3 O 10 •3H 2 O nanowires.G3: pure Ag 2−x (NH 4 ) x Mo 3 O 10 •3H 2 O nanowires.Staphylococcus aureus bacteria had been sufficiently eliminated with the presence of the nanowires.In G2, the nanowires originally seen on the N.A. surface disappeared gradually after 6-18 hours.In G3, the nanowires kept their original shapes.
Group 1 (G1) images show Staphylococcus aureus bacteria cultured on a base of N.A. at 40 • C for 18 hours.Group 2 (G2) images show Staphylococcus aureus bacteria mixed with dry Ag 2−x (NH 4 ) x Mo 3 O 10 •3H 2 O nanowires cultured on the same N.A. base at 40 • C for 18 hours.For comparison, Group 3 (G3) images show pure Ag 2−x (NH 4 ) x Mo 3 O 10 •3H 2 O nanowires on N.A. experimented under the same condition.The Staphylococcus aureus bacteria have been sufficiently eliminated with the presence of the nanowires.We note that, in G2, the nanowires originally seen on the N.A. surface disappeared gradually