Structural and Antibacterial Properties of Silver Helical Pentagon and L-shaped Nano Sculptured Thin Films

INTRODUCTION
Metallic nanostructures have attracted much attention from scientists due to their numerous applications in many areas of physical sciences, chemical sciences, and life sciences. By controlling the shape and dimensions of the nanostructures, surface chemistry, optical properties, and their biocidal activities can be managed. For example, experiments have demonstrated that the plasmon peak depends strongly on the morphology and assembly of the nanoparticles, such as diameter, aspect ratio, and shape [1, 2]. As reported in many studies, the antibacterial activity of metal nanostructures is mainly dependent on their size and shape. for example, Wigginton and coworkers [3], reported that silver nanoparticles attach to the Triptophanase (about 5 nm) in E. coli alter its enzymatic activity and distort its structure [4]. Size-dependent interaction of metal nanostructures with gram-negative bacteria has been widely reported, however little is known about the shape dependence of the antibacterial activity of metal nanostructures. Pal et al [5-7] reported that truncated triangular silver nanoplates had a stronger antibacterial activity against E. coli than other shapes such as spherical, rod-shaped nanoparticles. It may be anticipated that a bacterial cell in contact with silver nanoparticles takes in silver ions, which interdicts a respiratory enzyme(s), so cell is damaged. The antibacterial activity of the silver nanoparticles was analyzed and it was found that there was an inverse correlation between the nanostructures’ size and their biocidal activity, small particles exhibited higher antimicrobial activity than big particles due to their larger surface-to-volume ratios [8-10]. Reports on the mechanism of inhibitory action of silver ions on microorganisms showed that upon Ag+ treatment, DNA loses its replication ability, and the expression of ribosomal subunit proteins, as well as some other cellular proteins and enzymes essential to ATP production, become inactivated [11-13]. Also, studies have demonstrated that the reactivity of silver is favored by high-atom-density [14] facets with the bacterial surface [15].
Many scientists use the oblique angle deposition (OAD) method to produce dimensional nanostructures with a high surface area to volume ratio. Glancing angle deposition (GLAD) is another shape of the OAD where substrate rotation is also used. OAD and GLAD [16,17] techniques enable the production of various nanostructures like zig-zags, chevrons, nano-spirals, inclined columns, and branched nano-columns [18-22] for applications of sensing, energy, catalysis, and biomedicine [23].
In this work, we have used this technique and produced silver L-shaped and helical pentagon (HP) sculptured thin films and investigated their structural and antibacterial properties.

MATERIALS AND METHODS
 Silver (99.99% purity) L- and HP-shaped nano sculptured thin films were deposited at 75◦ oblique angle deposition on glass (15 × 15mm2 microscope slide) substrates, using resistive evaporation from tungsten boats with an outlet of 6mm diameter at room temperature. An Edwards (Edwards E19 A3) coating plant with a base pressure of 3 × 10−7 mbar and a deposition rate of 2.5A◦ s−1 was used. the vapor source (6mm in diameter) behaves like a point source and a 30 cm distance between the evaporation source and the substrate was chosen so a cosine distribution and straight trajectories of the vapor were achieved (i.e., no appreciable scattering due to the large mean free path (∼103–104 cm) occurs). Optical reflection from the reproduced samples was agreed to be within 5%. Different mechanical movements of substrate holders are controlled via an interface to a computer in which the related software is written and installed.
The structure was produced from an initial substrate position for the formation of one arm of the L or HP and then the process were repeated by successive rotation of the substrate holder by 180◦ for one time and 72◦ for ten times for the respective formation of the silver  L and HP structures’ arms in the anticlockwise direction. The length of each deposited arm is arranged to be about 635 nm and 350 nm for L and HP structures respectively. Considering that the deposition of the films was at 75◦ to the substrate surface normal, using the Tait rule, the height of each arm can be calculated as 635 × sin(90 – 53.2) = 380 nm for the L- shape and 350× sin(90-53.2)=209 nm for the HP-shape. Therefore, the total height of the L shape structure produced in this work with two arms should be equal to ∼760 nanometers and ~2.1 micrometers for an HP-shaped structure with ten arms, This is very close to the measurement from the cross-section of the films in Figure 1. All substrates were ultrasonically cleaned in heated acetone and then ethanol before being mounted on the substrate holder for deposition.
The deposition rate was measured by a quartz crystal deposition rate controller (Sigma Instruments, SQM-160, USA) positioned at almost the same azimuthal angle as that of the substrate and close to that. Field emission electron microscope (FESEM) samples were coated with a very thin layer of gold to prevent the charging effect (FESEM; Hitachi S-4100 SEM, Japan). The surface physical morphology and roughness was obtained by means of atomic force microscope (AFM; NT-MDT SOLVER, with a Si tip of 10nm radius in contact mode) analysis.
Antibacterial activity of the silver nano structures against the Escherichia coli ATCC 8739 (E.coli), Pseudomonas aeruginosa ATCC 10145 (Ps.aer), staphylococcus aureus ATCC 25923 (S.aureus), Micrococcus luteus ATCC 4698 (M.luteus ) and Candida albicans  PTCC 5027 (C.albicans) were studied using the so-called diffusion assay method. Among the bacteria studied, E coli and Ps.aer are gram negative, whereas S.aur and M.loteus are gram positive and C.albicans is a fungal genera [23]. Before experimenting, all films were cleaned with ethanol and acetone and autoclaved at 120 ºC for 15 min. Then they were placed into a sterilized Petri dish and irradiated by a UVC lamp for 30 minutes. The microorganisms were cultured on a nutrient agar plate and incubated at 37 ºC for 24 h, then added in 10 ml saline solution to get the concentration of bacteria 108 colony-forming units per milliliter (CFU/ml) corresponding to the MacFarland scale. 100 μl of the saline solution containing the microorganisms was added to the nutrient agar plate and spread with a sterile cotton swab. Then for the antibacterial test, each thin film was placed onto a cultured nutrient agar plate and incubated at 37 ºC for 24 h. After 24 hours area of inhibition zone of growth was measured.

RESULTS AND DISCUSSIONS
FESEM, AFM and XRD
Fig. 1, shows FESEM images of the surface structure and cross-section of L- and HP-shaped sculptured thin films. Cross-section of the films was obtained by fracturing the substrate that caused the braking of their arms. Hence, in Figure 1(b,d) a proper cross-section of  L- and HP-shaped thin films may not be clearly distinguished. The symmetry of the grown L- and HP- shaped on the surface of the films is shown by geometrical shapes (i.e., L shape or HP shape) while the orientation of grown arms is also shown by black arrows. These arrows show the principal axes that the columns grow along their directions. With regard to this observation, we should also point out the high surface diffusion of silver ad atoms because of the rise of substrate temperature from its initial setting during almost 5 hours of deposition, and that these films were grown on bare (unseeded) glass substrates. Therefore, all these parameters/effects prevent the L- and HP-shaped thin films from growing in a perfectly ordered structure. However, we may claim that these structures are more applicable for industrial or applied works, as preseeding is a costly procedure.
In Fig. 2, 2D and 3D AFM images of silver L- and HP-shaped sculptured thin films are given. The size distributions of the grains of these sculptured thin films were obtained from the 2D AFM images using JMicroVision Code, and the mean and the root mean square (RMS) surface roughness as well as, the average grain sizes are tabulated in Table 1. Fig. 3, shows the XRD patterns of the samples.  Their crystalline structure is consistent with the JCPDS card No: 04-0783. Crystallite size (coherently diffracting domains), D, was obtained by applying the Scherrer formula [24] to measure the full width at half maximum (FWHM) of the dominant peak of silver sculptured thin films (i.e., Ag(111)). The results with 10% accuracy are given in Table 1.

Antibacterial property of Ag L- and HP-shaped sculptured thin films
Fig. 4, shows the typical zone of inhibition (ZOI) for L-shaped (a) and helical pentagon (b) thin films on a Petri dish for E. coli ATCC 8739 bacteria, after 24 hours of incubation. ZOI was recorded as the zone of inhibition of the sample (see FIG.4). In Table 2, the ZOI obtained for silver L- and HP-shaped thin films against different test strains, E. coli ATCC 8739, S. aur ATCC 25923 , C. albicance PTCC 5027 and M. loteus ATCC 4698, are compared with each other and with other previously papers [25-28]. It can be concluded that in all of the bacteria cases, the helical pentagon thin films’ antibacterial effect dominates the other structure. This may be due to its higher intensity of (111) orientation, as discussed in Section 3.1, and higher surface-to-volume ratio due to the increased number of arms in this film. We also investigated the effect of these nanostructures on Ps.aer as a gram-negative bacteria (such as E.coli), results showed that two structures have the most antibacterial effect on this bacteria with the high ZOI of 3.90 cm for L-shaped thin film and 5.87 cm for helical pentagon thin film (nearly the all of the Petri dish), that they are the biggest ZOI in compare of previous papers. Hence, it may reiterate that by increasing the surface-to-volume ratio and surface roughness of thin films the probability of releasing silver atoms/ions in the medium increases as well as, the Ag(111) facet consists of the maximum number of atoms in the silver structure it should provide the highest antibacterial effect. Therefore, considering the structural and morphological results obtained for our HP- and L-shaped silver thin films, presented in the preceding sections, we expect that our nano-sculptured thin films should show an enhanced antibacterial property compared to a normal film (thin films deposited at a normal incident angle).

CONCLUSION
Sculptured thin film with HP-shaped shows higher antibacterial activity against all of the studied microorganisms than L-shape one. This may because of increasing the surface to volume ratio and surface roughness of this sculptured thin film as well as higher intensity of the Ag(111) orientation with the maximum number of atoms. so probability of releasing silver atoms/ions in the medium increases and the highest antibacterial activity is occured. These structures have the most antibacterial effect on the Pseudomonas aeruginosa ATCC 10145 bacteria, Their ZOI are 3 cm for L-shaped and 6.78 cm for HP-shaped sculptured thin films.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.