Enhanced Antibacterial Property of Sulfate-Doped Ag3PO4 Nanoparticles Supported on PAN Electrospun Nanofibers

Heterojunction nanofibers of PAN decorated with sulfate doped Ag3PO4 nanoparticles (SO42−-Ag3PO4/PAN electrospun nanofibers) were successfully fabricated by combining simple and versatile electrospinning technique with ion exchange reaction. The novel material possessing good flexibility could exhibit superior antibacterial property over sulfate undoped species (Ag3PO4/PAN electrospun nanofibers). FESEM, XRD, FTIR, XPS and DRS were applied to characterize the morphology, phase structure, bonding configuration, elemental composition, and optical properties of the as fabricated samples. FESEM characterization confirmed the successful incorporation of SO42−-Ag3PO4 nanoparticles on PAN electrospun nanofibers. The doping of SO42− ions into Ag3PO4 crystal lattice by replacing PO43− ions can provide sufficient electron-hole separation capability to the SO42−-Ag3PO4/PAN heterojunction to generate reactive oxygen species (ROS) under visible light irradiation and enhances its antibacterial performance. Finally, we hope this work may offer a new paradigm to design and fabricate other types of flexible self-supporting negative-ions-doped heterojunction nanofibers using electrospinning technique for bactericidal applications.

long lasting, and little drug resistance. Based on the action mechanism, inorganic materials are divided into semiconductor materials and metal-base materials. All semiconductor materials act as photocatalysts and are known to cause the formation of biologically reactive oxygen species (ROS), including hydroxyl/superoxide (OH • /O2 •− ) radicals in the presence of light. Thus, the produced ROS are responsible for the bactericidal activity of semiconductor materials but metal-based materials act as bactericidal agents without light assistance [7][8][9][10]. Among such semiconductor materials, silver phosphate (Ag 3 PO 4 ) has drawn considerable attention as an antibacterial semiconductor material in recent years [11,12]. It is a narrow band gap semiconductor (2.36 eV), having the capability to generate ROS under visible light irradiation; however, it is noted that the application of Ag 3 PO 4 as a photocatalyst is limited due to its poor chemical stability, when used without any sacrificial reagent in aqueous solution [13,14]. Furthermore, the agglomeration tendency of semiconductor photocatalyst when used in powder form is another serious issue, which reduces the effective surface area of particles, thereby decreasing their efficiency and making separation process more difficult after use. Therefore, after the breakthrough work carried out by Ye et al. [14], successive investigations have been performed to improve the photocatalytic efficiency of this semiconductor material with sufficient charge separation ability and stability by coupling it with other semiconductor/s [15,16], fabricating composites (with graphene [17,18], carbon nanotubes [19,20], non-metallic sorbent [21]) and doping suitable ions [22][23][24].
Semiconductor material doped with suitable ions could be an effective approach to design a photocatalyst with enhanced activity and stability [25]. Doping of suitable ions into the crystal lattice of semiconductor material provides significant capability to prevent the recombination of photogenerated electron-hole pairs and consequently improves the photocatalytic stability of the semiconductor materials. On that note, various attempts have been made to increase the photocatalytic performance of Ag 3 PO 4 through cations doping into Ag 3 PO 4 crystal lattice [26,27]. But the investigation on antibacterial activity of Ag 3 PO 4 doped with suitable anions has been rarely reported. However, recently published report demonstrated that sulfur-doped Ag 3 PO 4 can improve its photocatalytic activity on the basis of hybrid density-functional calculation [28] but the doping of sulfur into Ag 3 PO 4 crystal lattice is not so easy by post-treatment process due to the strong P-O bond. Consequently, SO 4 2− might be a suitable doping ion into Ag 3 PO 4 crystal lattice by replacing PO 4 3− since SO 4 2− has smaller radius (0.218 nm) than that of PO 4 3− (0.230 nm) [29,30]. The use of polymer electrospun nanofibers for incorporating photocatalyst nanoparticles through different fabrication methods is a promising strategy to provide enough reactive sites and avoid nanoparticles from being wasted during the separation process [31][32][33][34]. Because of extra-long one-dimensional structures, nanosized diameters and good flexibility, polymer electrospun nanofibers are considered to be good supports for immobilizing nanoparticles [35,36]. PAN is a widely used polymer for the fabrication of nanofibers using simple and versatile electrospinning techniques because of its good processability, environmental stability and low density. Therefore, PAN nanofibers are being used extensively as supports for photocatalyst nanoparticles [32,37]. Hence, to understand the combination of respective properties of SO

Results and Discussion
The crystalline structure and effect of SO 4 2− doping on Ag 3 PO 4 nanoparticles were investigated by XRD (Figure 1a).   The morphological structure and distribution of nanoparticles on the surface of nanofibers were studied using FESEM ( Figure 2). All formulations possess bead-free, continuous and randomly oriented nanofibers having an average diameter of 425 nm. Compared to Na 2 HPO 4 /PAN electrospun nanofibers (Figure 2a), the surface of AP/PAN nanofibers ( Figure 2b) and S-AP/PAN nanofibers (Figure 2c) was no longer smooth due to the presence of secondary nanoparticles uniformly immobilized across the PAN nanofibers surface with some agglomerations. Figure 2d represents the high magnification image of S-AP/PAN showing the fiber/particles interface. As depicted in the figure, all the nanoparticles are well adhered to the nanofibers, so that these particles will not fall off the nanofibers in operational conditions. Furthermore, a cross section FESEM image was employed to measure the thickness of the nanofibers mat ( Figure 2e). It appeared that the thickness of the S-AP/PAN nanofibers mat was estimated to be around 38 µm. The insets (Figure 2a [40]. In AP/PAN and S-AP/PAN, absorption bands located at about 550 cm −1 and 981 cm −1 could be assigned to the molecular vibration of PO 4 3− [37,41].   The optical band gap energies (E g ) of S-AP/PAN and AP/PAN were estimated using a plot of (αhν) 1/2 versus energy (hν) [44]. As depicted in Figure 5b, the band gap energies of S-AP/PAN and AP/PAN were estimated to be 2.74 eV and 2.72 eV, respectively. This indicated that the band gap energy was increased slightly with SO 4 2− doping. The elemental composition and chemical state of elements in AP/PAN and S-AP/PAN were analyzed by XPS ( Figure 6). The noticeable peaks of P 2p, C 1s, Ag 3d, N 1s and O 1s were clearly detected in the survey spectra of both samples (Figure 6a,b). The characteristics peaks corresponding to C 1s and N 1s which appeared around 284.6 eV and 397.6 eV, respectively, were from PAN ( Figure 6a) [45]. Additionally, information regarding the specific nature of S, P, Ag and O was obtained from high resolution XPS spectra of both samples. In Figure 6c, the peak appeared at around 168.37 eV in high resolution spectrum of S 2p in S-AP/PAN is attributed to S 6+ [46]. This result further confirmed that the incorporation of SO 4 2− into the Ag 3 PO 4 crystal lattice could occur during the synthesis process.
The binding energies corresponding to P 2p and Ag 3d peaks of S-AP/PAN were found shifted to higher values compared to that of AP/PAN (Figure 6d,e). Such shifting might happen due to the doping of SO 4 2− , which results in decreased electron density around P and Ag because of higher electronegativity of S [47]. Similar behavior was observed for O 1s peak of S-AP/PAN compared to that of AP/PAN ( Figure 6f). Therefore, all the results from XPS analysis suggested the incorporation of S in the form of SO 4 2− in Ag 3 PO 4 crystal lattice due to its strong interaction with the rest of the elements [25].
The antibacterial activities of pure PAN, AP/PAN and S-AP/PAN samples against E. coli and S. aureus were assessed by determining the diameter of inhibition zones, where pure PAN was used as a control. Antibacterial ability in the form of inhibition zones evaluated by the disk diffusion assay is shown in Figure 7. As shown in the figure, pure PAN showed no zone of inhibition indicating the lack of antibacterial activity for both Gram negative and Gram positive bacteria, while AP/PAN and S-AP/PAN clearly showed zones of inhibition. The zones of inhibition were 8.1 ± 1.52 mm and 9.7 ± 1.15 mm for E. coli for AP/PAN and S-AP/PAN, respectively (Figure 7a,b,g). Similarly, the zones of inhibition were 7.5 ± 0.57 mm and 8.9 ± 0.5 mm for S. aureus for AP/PAN and S-AP/PAN, respectively (Figure 7c,d,h). Figure 7e,f represent disk diffusion test on E. coli and S. aureus, respectively, using pure PAN and S-AP/PAN in dark condition. As depicted, no distinct zone of inhibition was observed, which signifies the absence of photogenerated ROS in dark condition. Furthermore, the antibacterial ability of pure PAN, AP/PAN and S-AP/PAN samples was tested by observing the survival of E. coli and S. aureus cells under day light (Figure 8). In this test, pure PAN was also used as control, which did not show any remarkable antibacterial activity but from the figure it is clear that S-AP/PAN had the lowest survivability of both types of bacteria compared to AP/PAN, however AP/PAN and S-AP/PAN showed significant levels of bacterial growth suppression activity compared to Pure PAN. Therefore, from these results it was seen that S-AP/PAN exhibited higher bacterial growth suppressing activity in both the methods and it can offer great promise to its application in the bactericidal activity. Moreover, the higher antibacterial efficacy of S-AP/PAN than that of S/PAN against E. coli compared to S. aureus might be related the cellular wall content differences between Gram-negative and Gram-positive bacteria [48,49].
The enhanced antibacterial performance of SO

Fabrication of Na 2 HPO 4 /PAN Nanofibers
First, 0.46 g of Na 2 HPO 4 powder was ground with a mortar and pestle and dispersed in 14 ml of DMF using ultrasonication for 1 h. Then 1.5 g of PAN powder was added to the above dispersion with continuous magnetic stirring for 12 h in order to obtain homogeneous solution for electrospinning. Electrospinning of the prepared solution was carried out by loading into a plastic syringe provided with plastic micro-tip and using high voltage power supply at an applied voltage of 18 kV adjusting tip to collector distance 12 cm. The developing nanofibers were collected on a drum collector rotated at a constant speed by DC motor. All the experimental works were conducted at room temperature and atmospheric pressure. Afterward, the resulting electrospun nanofibers were vacuum dried at 70 • C for 12 h to remove the residual solvent.

Characterization
Information about phase and crystallinity of the samples were investigated using X-ray diffractometer (XRD, Empyrean, PANAlytical, Netherlands) with Cu Kα (λ = 1.540 Å) radiation over Bragg angles ranging from 10 • to 80 • . Morphological structure and distribution of nanoparticles on the surface of nanofibers were characterized using field emission scanning electron microscope (FESEM, GeminiSEM 500, Zeiss, UK) equipped with energy dispersive X-ray spectroscopy (EDS) to analyze the elemental composition of samples. Prior to characterization, samples were prepared by coating with platinum for 180 s using a Pt-coater (E-1030, Hitachi). Bonding configuration of the polymer with nanoparticles was characterized with the help of Fourier-transform infrared (FT-IR, FT/IR-4200, Jasco international Co., Ltd.) using attenuated total reflectance mode (ATR). Light absorption behavior of all samples was investigated from Uv-vis diffusive reflectance spectra (DSR) obtained from UV-vis spectrophotometer (Lambda 900, PerkinElmer, USA). Furthermore, the surface element composition analysis of SO 4 2− -doped/undoped samples was recorded using X-ray photoelectron spectroscopy (XPS, VG scientific Co., ESCA LAB MK II).

Antibacterial Activity Investigation
The antibacterial efficacy of the samples was assessed by using Gram negative E. coli (ATCC 25922) and Gram positive S. aureus (ATCC 4330) bacterial strains. Both disk diffusion and bacterial colony count tests were performed to determine growth suppressing action of pure PAN, AP/PAN and S-AP/PAN under day light conditions. In the disk diffusion test, Tryptic soya agar was used to perform the disk diffusion method to evaluate the antibacterial activity of the samples according to the previously described procedure [54]. Individual 0.5 McFarland sterile normal saline suspensions were prepared from freshly grown E. coli and S. aureus culture. A 100 µl cell suspension was plated in each tested petri dish and 5 mm disk of every samples were carefully dropped on top of the culture plates. All petri dishes were incubated at 37 • C for 12 h under day light and after incubation the zone of inhibitions were measured using ImageJ software. For comparison, the disk diffusion test was performed to evaluate the antibacterial activity of pure PAN and S-AP/PAN towards E. coli and S. aureus in dark conditions. The antibacterial effects of Pure PAN, AP/PAN and S-AP/PAN were further determined via bacterial colony count test on tryptic soya agar plates according to previous method with modification [55]. The fragments (3 cm × 5 cm) of test samples were embedded at the bottom of petri dish while making agar plates. A 0.5 McFarland cell suspension was serially diluted (10-fold dilution) 4 times in saline solution and, 100 µl aliquots of E. coli and S. aureus cell suspension were spread on each tested petri dish. Culture dishes were incubated at 37 • C for 12 h and the bacterial colonies were counted after incubation. All experiments were performed in triplicates, and statistical analysis was carried out using SPSS software.

Statistical Analysis
All statistical data were expressed as the mean ± s. d., as indicated. Statistical significance was performed by one-way ANOVA using SPSS software [56]. p < 0.05 was considered as significant and represented as * p < 0.05, ** p < 0.01 and *** p < 0.001.

Conclusions
This was the first time that a heterojunction of sulfate-doped Ag 3 PO 4 nanoparticles supported on PAN electrospun nanofibers was fabricated using the electrospinning technique followed by the ion exchange method. The antibacterial activity of the fabricated material was investigated on E. coli