Zn and Zn-Fe nanostructures with multifunctional properties as components for food packaging materials

Citation: Lamsaf, H.; Ballesteros, L.F.; Cerqueira, M.A.; Teixeira, J.A.; Pastrana, L.M.; Rebouta, L.; Carvalho, S.; Calderon, S. Zn and Zn-Fe Nanostructures with Multifunctional Properties as Components for Food Packaging Materials. Nanomaterials 2022,12, 2104. https://doi.org/ 10.3390/nano12122104 Academic Editor: Krasimir Vasilev Received: 31 May 2022 Accepted: 15 June 2022 Published: 18 June 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). nanomaterials Article Zn and Zn-Fe Nanostructures with Multifunctional Properties as Components for Food Packaging Materials Hafsae Lamsaf 1,2 , Lina F. Ballesteros 3,4, Miguel A. Cerqueira 2, JoséA. Teixeira 3,4 , Lorenzo M. Pastrana 2, Luís Rebouta 1, Sandra Carvalho 5and Sebastian Calderon 1,2,* 1CF-UM-UP, Centre of Physics of Minho and Porto Universities, Campus of Azurém, 4800-058 Guimarães, Portugal; [email protected] (H.L.); [email protected] (L.R.) 2 INL—International Iberian Nanotechnology Laboratory, Av. Mestre JoséVeiga s/n, 4715-330 Braga, Portugal; [email protected] (M.A.C.); lor[email protected] (L.M.P.) 3CEB—Centre of Biological Engineering, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal; [email protected] (L.F.B.); [email protected] (J.A.T.) 4LABBELS–Associate Laboratory, Braga/Guimarães, Portugal 5CEMMPRE, Mechanical Engineering Department, University of Coimbra, 3030-788 Coimbra, Portugal; [email protected] *Correspondence: [email protected] Abstract: Metallic and bimetallic nanostructures have shown interesting chromatic and antibacterial properties, and they can be used in various applications. In this work, zinc (Zn) and iron (Fe) nanostructures were produced with different morphologies: (i) pure Zn; (ii) Zn-Fe nanoalloys; (iii) Zn-Fe nanolayers (Zn-Fe NLs); and (iv) Zn nanolayers combined with Fe nanoparticles ( Zn NLs + Fe NPs ). The aim was to produce components for food packaging materials with active and intelligent properties, including oxygen absorption capacity, chromatic properties, and antibacterial properties. Thus, the morphology, structure, and chemical composition of the samples were characterized and correlated with their oxidation, chromatic, and antibacterial properties. The results revealed a relevant reduction in the coating’s opacity after oxidation varying from 100 to 10% depending on the morphology of the system. All coatings exhibited significant antibacterial activity against S. aureus, revealing a direct correlation with Zn content. The incorporation of Fe for all atomic arrangements showed a negative impact on the antibacterial effect against E. coli, decreasing to less than half the zone of inhibition for Zn-Fe NLs and Zn NLs + Fe NPs and suppressing the antibacterial effect for Zn-Fe alloy when compared with the pure Zn system. Keywords: zinc–iron; nanostructure; sputtering; food packaging; chromatic effect; antibacterial activity 1. Introduction The preservation of food quality and the extension of its shelf life have become the main subject of many investigations in the field of packaging materials. Consequently, food packaging manufacturers have developed multifunctional materials that can guarantee food quality, shelf-life extension, cost efficiency, product safety, and consumer demand. Thus, to achieve such multifunctional requirements, researchers and industries have focused on the development of novel nanomaterials compatible with food products. Some examples of these are metallic nanoparticles, which have been explored as antibacterial agents [ 1 – 3 ], antioxidants [4–6], catalyzers [7,8], and photocatalytic and scavenging mediators [3]. Nanoparticles (NPs) of transition metals have demonstrated potential in technological applications compared with macroscopic materials, particularly when used in food packaging, enhancing the mechanical properties and controlling the biodegradability of the produced materials [ 3 , 9 , 10 ]. Moreover, studies have confirmed the benefit that such NPs bring to consumers, since the NPs contribute to extending the shelf life of food, ensuring better traceability and providing reinforced protection [ 11 , 12 ]. Several metals, such as Nanomaterials 2022,12, 2104. https://doi.org/10.3390/nano12122104 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2022,12, 2104 2 of 14 zinc (Zn), titanium (Ti), iron (Fe), and copper (Cu), have been used to produce NPs for food packaging materials [ 13 , 14 ], acting as antimicrobial agents, pigments, and oxygen scavengers [1,15,16]. Zinc oxide (ZnO) NPs, known for their multifunctional effects, are currently being studied in food packaging materials as antibacterial agents, for preventing food contamination due to harmful bacteria [ 1 ], and as absorbers of ultraviolet light (UV), taking advantage of the wide bandgap (Eg = 3.37 eV) of ZnO [ 17 ]. Shankar et al. [ 18 ], for instance, showed an increase in minced fish cake shelf life and strong antibacterial activity against foodborne pathogenic bacteria, Escherichia coli, and Listeria monocytogenes for poly (lactic acid) and ZnO (PLA/ZnO) NP composite films. Moreover, in previous research by the authors of the current study, Zn NPs in the metallic state demonstrated oxygen scavenging properties at high humidity, useful for food packaging applications [ 19 ]. Fe, on the other hand, is a known colorant in food [ 20 ] and has been used in nanopowder form as an oxygen scavenger in lowand high-relative humidity environments, with a scavenging rate three times higher than that of microscavengers when exposed at 100% relative humidity [21]. Bimetallic nanostructures have been shown to enhance the functionality of metallic NPs, providing not only the individual properties of the components but synergetic new phenomenology due to physical binding between the metals. Consequently, combining Zn and Fe metallic nanostructures was expected to provide multifunctional characteristics to food packaging materials. Fe-Zn oxide, for example, has demonstrated good magnetic and antibacterial properties, depending on its composition and morphology. Gordon et al. [ 22 ] showed that higher ratios of Zn/Fe NPs had more important antibacterial activity against Staphylococcus aureus and Escherichia coli. Furthermore, recent work on galvanic oxidation of bimetallic Zn-Fe NPs for oxygen scavenging [ 23 ] revealed that the bimetallic system of Zn-Fe accelerated the oxidation mechanism. The obtained material presented great potential to be used as an oxygen scavenger [ 23 ], as precise control of the morphology could be obtained by using magnetron sputtering techniques [24]. The morphology of NPs is another factor that strongly influences their multifunctional properties, and therefore, a convenient production method for NPs needs to be chosen. NPs are usually prepared using physical, chemical, and biological methods [ 25 – 27 ], which can influence their geometry, aspect ratio, and distribution [ 3 , 28 , 29 ] as well as their composition and toxicity. However, the incorporation of the nanoparticles into packaging materials is a nontrivial process, dramatically reducing the performance of the nanostructures because of agglomeration [ 30 ]. Thus, direct production of nanostructures on packaging materials is desirable. Magnetron sputtering is a candidate method to achieve this, since it is commercially used in packaging materials and allows precise control of the morphology and composition of the nanostructures, reducing the use of toxic chemicals during processing [19,31]. In the present work, four different coating systems with dissimilar atomic arrangements, namely pure Zn, Zn-Fe alloy, Zn-Fe nanolayers (Zn-Fe NLs), and Zn nanolayers containing Fe nanoparticles (Zn NLs + Fe NPs), were produced. Conventional magnetron sputtering and hybrid magnetron sputtering coupled to a cluster gun were used to control the morphology, structure, and chemical composition of the coatings, which were later correlated with the coatings’ antibacterial and chromatic properties. It was demonstrated that the design of the coatings’ architecture allowed control of the oxidation of the Zn-Fe nanostructures, leading to controllable changes in their chromatic and antibacterial properties. Finally, the system with the best functionalities to be used in food packaging materials was established. 2. Materials and Methods 2.1. Production of the Materials The nanostructures were produced using two different sputtering processes to adjust the atomic arrangements between the Zn and Fe, as shown in Figure 1. Classical magnetron sputtering was used to produce pure Zn, Zn-Fe alloy, and Zn-Fe nanolayers (Zn-Fe NLs) coatings as shown in Figure 1a, b, d, respectively. On the other hand, a hybrid Nanomaterials 2022,12, 2104 3 of 14 magnetron sputtering with a cluster gun was used to produce the Zn nanolayers containing Fe nanoparticles (Zn NL + Fe NPs) coating system, as shown in Figure 1c. Nanomaterials 2022, 12, x FOR PEER REVIEW 3 of 15 2. Materials and Methods 2.1. Production of the Materials The nanostructures were produced using two different sputtering processes to adjust the atomic arrangements between the Zn and Fe, as shown in Figure 1. Classical magnetron sputtering was used to produce pure Zn, Zn-Fe alloy, and Zn-Fe nanolayers (Zn-Fe NLs) coatings as shown in Figure 1a, b, d, respectively. On the other hand, a hybrid magnetron sputtering with a cluster gun was used to produce the Zn nanolayers containing Fe nanoparticles (Zn NL + Fe NPs) coating system, as shown in Figure 1c. Figure 1. Diagram of the different morphologies produced by classical magnetron sputtering and hybrid magnetron sputtering with a cluster gun: (a) pure Zn; (b) Zn-Fe alloy; (c) Zn NLs + Fe NPs; and (d) Zn-Fe NLs. 2.1.1. Classical Magnetron Sputtering Figure 2a presents a top-view layout of the classical chamber where the samples were produced. The nanostructures were deposited using a DC-pulsed magnetron sputtering technique with two 200 × 100 mm 2 high-purity targets of Zn and Fe located 180° from each other (Zn TRG, 99.99%, Fe TRG, 99.95%, acquired from Testbourne Ltd., Basingstoke, UK). The chamber was evacuated at an initial pressure of 2 × 10 −4 Pa, then set up to a working pressure of 5 Pa by introducing argon (ΦAr = 80 sccm). The substrates, rotating at a constant speed of 8 rpm, were maintained at a constant temperature between 300 and 313 K during the deposition. Figure 1. Diagram of the different morphologies produced by classical magnetron sputtering and hybrid magnetron sputtering with a cluster gun: ( a ) pure Zn; ( b ) Zn-Fe alloy; ( c ) Zn NLs + Fe NPs; and (d) Zn-Fe NLs. 2.1.1. Classical Magnetron Sputtering Figure 2a presents a top-view layout of the classical chamber where the samples were produced. The nanostructures were deposited using a DC-pulsed magnetron sputtering technique with two 200 × 100 mm 2 high-purity targets of Zn and Fe located 180 ◦ from each other (Zn TRG, 99.99%, Fe TRG, 99.95%, acquired from Testbourne Ltd., Basingstoke, UK). The chamber was evacuated at an initial pressure of 2 × 10 −4 Pa, then set up to a working pressure of 5 Pa by introducing argon ( Φ Ar = 80 sccm). The substrates, rotating at a constant speed of 8 rpm, were maintained at a constant temperature between 300 and 313 K during the deposition. Nanomaterials 2022, 12, x FOR PEER REVIEW 4 of 15 Figure 2. (a) Top-view layout for classical magnetron sputtering; (b) Zn and Fe target ON/OFF state during classical magnetron deposition; (c) top-view layout for hybrid magnetron sputtering with a cluster gun; and (d) Zn and Fe target ON/OFF state during hybrid deposition. The deposition parameters are presented in Table 1. Because of the low Fe deposition rate, the process was performed by turning the Zn target ON/OFF while the Fe target was always maintained at ON (Figure 2b), which allowed for better control of the composition of the coatings. Zn-Fe alloy coatings, for instance, were deposited in 15 thin layers to ensure the alloy morphology by distributing Zn and Fe over the entire surface, while for Zn-Fe NLs, the Zn target was turned OFF only at the end of the Zn layer deposition to allow the bilayer morphology. Table 1. Deposition parameters to produce the different coating systems. Process Coating J Zn (mA/cm 2 ) J Fe (mA/cm 2 ) Zn + Fe Layer Time (min) Fe Layer Time (min) N. of Layers Classical Pure Zn 0.5 — 14.5 * 1 Classical Zn-Fe alloy 0.5 2.5 1 4.17 15 Cluster Zn NLs + Fe NPs 1.9 3.2 0.5* 10 24 Classical Zn-Fe NLs 1.0 2.5 15 10 1 * only Zinc target is active; J: current density. 2.1.2. Hybrid Magnetron Sputtering—Cluster Gun Figure 2c shows the scheme of the chamber setup where the Zn NL + Fe NP deposition was performed. It was divided into two parts: (i) the main chamber, where the Zn target (diameter 50.8 mm, thickness 4.5 mm, and purity: 99.9%, purchased from Testbourne Ltd., Basingstoke, UK) was placed at 6 cm from the substrate, and (ii) the gun chamber, where the Fe target (diameter 6.9 cm, thickness 3 cm, purity 99.95% obtained from Testbourne Ltd., Basingstoke, UK) was located. The gun chamber contained a DC magnetron sputtering cluster source and was connected to a water-cooling system. This chamber had two apertures with diameters of 2.5 mm and 4 mm, respectively, to guarantee that the flow direction of the cluster beamed toward the main chamber because of Figure 2. ( a ) Top-view layout for classical magnetron sputtering; ( b ) Zn and Fe target ON/OFF state during classical magnetron deposition; ( c ) top-view layout for hybrid magnetron sputtering with a cluster gun; and (d) Zn and Fe target ON/OFF state during hybrid deposition. Nanomaterials 2022,12, 2104 4 of 14 The deposition parameters are presented in Table 1. Because of the low Fe deposition rate, the process was performed by turning the Zn target ON/OFF while the Fe target was always maintained at ON (Figure 2b), which allowed for better control of the composition of the coatings. Zn-Fe alloy coatings, for instance, were deposited in 15 thin layers to ensure the alloy morphology by distributing Zn and Fe over the entire surface, while for Zn-Fe NLs, the Zn target was turned OFF only at the end of the Zn layer deposition to allow the bilayer morphology. Table 1. Deposition parameters to produce the different coating systems. Process Coating JZn (mA/cm2) JFe (mA/cm2) Zn + Fe Layer Time (min) Fe Layer Time (min) N. of Layers Classical Pure Zn 0.5 — 14.5 * 1 Classical Zn-Fe alloy 0.5 2.5 1 4.17 15 Cluster Zn NLs + Fe NPs 1.9 3.2 0.5* 10 24 Classical Zn-Fe NLs 1.0 2.5 15 10 1 *only Zinc target is active; J: current density. 2.1.2. Hybrid Magnetron Sputtering—Cluster Gun Figure 2c shows the scheme of the chamber setup where the Zn NL + Fe NP deposition was performed. It was divided into two parts: (i) the main chamber, where the Zn target (diameter 50.8 mm, thickness 4.5 mm, and purity: 99.9%, purchased from Testbourne Ltd., Basingstoke, UK) was placed at 6 cm from the substrate, and (ii) the gun chamber, where the Fe target (diameter 6.9 cm, thickness 3 cm, purity 99.95% obtained from Testbourne Ltd., Basingstoke, UK) was located. The gun chamber contained a DC magnetron sputtering cluster source and was connected to a water-cooling system. This chamber had two apertures with diameters of 2.5 mm and 4 mm, respectively, to guarantee that the flow direction of the cluster beamed toward the main chamber because of pressure differences. The substrate holder was located 10 cm from the aperture, while the Fe magnetron was 8 cm away from the large nozzle. Ar was used as a sputtering gas with the flow ΦAr = 60 sccm , resulting in working pressures of 88 Pa and 0.4 Pa in the cluster source and the main chamber, respectively. The morphology of the Zn NLs + Fe NPs (Figure 1c) was produced using alternate deposition by turning ON/OFF the power applied to each magnetron (as shown in Figure 2d). This multilayer system allowed for precise control of the content of Fe because of the lower deposition rate of Fe in the cluster gun compared with the conventional magnetron sputtering used for Zn deposition. An example of the final coating All coatings were deposited onto Si-wafers (supplied by Siegert Wafer GmbH, Aachen, Germany), TEM Cu-grids with ultrathin carbon layers (400 mesh, obtained from Monocomp Instrumentación S.A., Madrid, Spain), transparent glass slides (purchased from Fisher Scientific, Leicestershire, UK), and poly L lactic acid (PLA) biopolymer films (50 µ m thickness, acquired from Goodfellow GmbH, Hamburg, Germany). The Si and glass substrates were sequentially cleaned with distilled water, acetone, and 95% ethanol (10 min with each solvent) in an ultrasonic bath to remove impurities on the surface, while the PLA and the Cu-grids were placed as bought because of their sensitivity to solvents. 2.2. Methodology 2.2.1. Morphology, Composition, and Structure Scanning electron microscopy (SEM) images of the coatings deposited on Si-wafers were performed with an FEI Helios NanoLab 450S Dual Beam (Eindhoven, The Netherlands ) with a through-the-lens detector (TLD), operating at 5 keV with a beam current of 0.4 nA . High angle annular dark-field (HAADF) images obtained by scanning transmission electron microscopy (STEM) were collected from samples deposited on ultrathin carbon grids at 200 keV on a double corrected FEI Titan Themis (Eindhoven, The Netherlands). Furthermore, energy-dispersive X-ray spectroscopy (EDS) mapping in transmission mode was Nanomaterials 2022,12, 2104 5 of 14 acquired using a double corrected FEI Titan Themis (Eindhoven, The Netherlands) operated at 200 keV and equipped with a Super-X EDS detector. To determine the elemental distribution, iterative maps of 512 × 512 pixels with a dwell time per pixel of 10 µ s at 200 keV were acquired. Inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to quantify the total concentration of Zn and Fe in each coating deposited on glass slide substrates. Thus, samples of approximately 1 cm 2 were placed in 15 mL Falcon tubes and immersed in 10 mL of 65% (v/v) HNO 3 . The samples were left at room temperature for 24 h and then diluted and filtrated through 0.22 µ m of regenerated cellulose membranes. The measurements were carried out in an Optima 8000 ICP-OES (Perkin Elmer, Boston, MA, USA), operating with an axial plasma view and 1400 W, at wavelengths of 214 and 238 nm for Zn and Fe, respectively. X-ray photoelectron spectroscopy (XPS) of the coatings deposited on PLA films was performed in a Thermo Scientific ESCALAB 250Xi (Eindhoven, The Netherlands) with the aim to determine the surface composition and chemical binding energies. The analysis was conducted at 15 kV (200 W) with a monochromatic Al K α X-ray source of 1486.7 eV. Data acquisition was carried out with a charge neutralization system and a pressure lower than 1×10−6Pa. Additionally, structural analysis was performed to identify the phases in the coatings. Selected area electron diffraction (SAED) patterns were obtained using samples deposited on ultrathin carbon grids. 2.2.2. Functional Properties Color parameters and the opacity of the coatings deposited on glass substrates were determined using a Minolta colorimeter (CR 400, Minolta, Japan). The equipment was calibrated with a white standard color plate, used as a background for color measurements (L*,a*,b*) according to Ballesteros et al. [ 32 ]. The opacity of the coatings, expressed in percentage (%), was calculated by the Hunter lab method, using the ratio of the opacity of each sample on a black standard (Yb) and a white standard (Yw), as described by Casariego et al. [ 33 ]. The results were simulated using Adobe Photoshop software (CS6, Adobe Inc. San Jose, CA, USA)). Five replicates of each coating were made for both color and opacity measurements, which were analyzed as deposited (1st day) and after being exposed at 98% relative humidity (7th day) using a saturated salt solution of K 2 SO 4 at room temperature. The antibacterial activity of the coating studied was performed against the Grampositive Staphylococcus aureus (EG17 strain) bacterium and Gram-negative Escherichia coli (CECT 736 strain) bacterium, obtained from the Centre of Biological Engineering collection of the University of Minho. The zone of inhibition (ZOI) test was carried out as described by the Clinical and Laboratory Standards Institute [ 34 ] in order to determine the diffusion in the agar of Zn and Fe from the PLA film’s surface, which was used as a substrate. The halo size was used to quantify the inhibition area of the coatings against bacterial growth. The bacteria were cultivated into 20 mL of nutrient broth (NB, Oxoid) and incubated at 37 ◦ C, 150 rpm for 18 h. The resultant cell suspension for each strain was adjusted to an optical density (80–82% in the McFarland standards) between 0.09 and 0.11, measured at 620 nm, indicating a concentration of 1 × 10 8 CFU/mL. Later, the inocula were diluted in NB to 1 × 10 6 CFU/mL, and then, aliquots of 200 µ L of cell suspensions were spread with sterile swabs on Petri dishes (90 mm) containing approximately 25 mL of nutrient agar (NA, Oxoid). The Zn and Fe coatings deposited on PLA films of approximately 0.5 ×1.5 mm2 were previously sterilized by exposing them to UV light for 1 h and subsequently placed in contact with the agar. The Petri dishes were incubated at 37 ◦ C for 24 h, and pure PLA films were used as a negative control. The transparent halo formed around the samples was evaluated for each bacterium to define the inhibition of bacterial growth. The ZOI was measured through the Image J-64 software (V1.52p, National Institute of Health, Bethesda, Nanomaterials 2022,12, 2104 6 of 14 MA, USA) and expressed in mm 2 . Each sample was evaluated in duplicate and repeated at least in two independent assays. 2.2.3. Statistical Analysis GraphPad Prism V6.1 by Dotmatics (San Diego, CA, USA) was used to carry out a one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test to evaluate the significant differences (p< 0.05) among the different coatings. 3. Results and Discussion 3.1. Morphology, Structure, and Chemical Composition Characterizations STEM-EDS chemical mapping was carried out to confirm the morphologies and element distribution of the Zn-Fe nanostructures, as shown in Figure 3. Both a top view and a cross-section were acquired. The results showed different Fe distributions depending on the production method. For comparison, a pure Zn nanostructure was shown, revealing a coating formed by large grains with random shapes (Figure 3a). The Zn-Fe alloy showed a homogeneous distribution, as depicted in Figure 3b. On the other hand, the Zn NL + Fe NP sample (Figure 3c) showed the presence of Fe NPs, which was due to the agglomeration process that occurred in the cluster gun. These NPs had a large size distribution, forming a separate phase that was easily identified. The Fe NPs varied between 5 and 23 nm, with an average size of 11 nm (see Figure S1 in Supplementary Materials). Finally, Figure 3d shows that the Zn-Fe NL sample had a bilayer morphology and a uniform distribution of Fe surrounding the Zn nanolayer. All the coatings exhibited passivated surface and column boundaries, as observed in the oxygen signal, in agreement with the XPS analysis, as later demonstrated. Nanomaterials 2022, 12, x FOR PEER REVIEW 7 of 15 Figure 3. STEM-EDS images of the coatings: (a) pure Zn; (b) Zn-Fe alloy; (c) Zn NLs + Fe NPs; and (d) Zn-Fe NLs, viewed from the top (left) and cross-section (right). The scale bar corresponds to 40 nm. The red, blue, and green colors in the figure correspond to Zn, Fe, and O signals, respectively. Lower magnification STEM top-view images and SEM cross-section images are shown in Figure 4. The results evidenced full coverage of the samples’ surface, but with significant differences in the morphology depending on the coatings’ architecture. All the coatings revealed a large distribution of grain sizes (Figure 4a–c), except for the Zn-Fe alloy, which showed a more compact morphology (Figure 4b) than all the other samples. Both the Zn NLs + Fe NPs and Zn-Fe NLs had heterogeneous particle distributions, but the latter showed a clear bimodal particle size distribution. Figure 3. STEM-EDS images of the coatings: ( a ) pure Zn; ( b ) Zn-Fe alloy; ( c ) Zn NLs + Fe NPs; and ( d ) Zn-Fe NLs, viewed from the top (left) and cross-section (right). The scale bar corresponds to 40 nm . The red, blue, and green colors in the figure correspond to Zn, Fe, and O signals, respectively. Lower magnification STEM top-view images and SEM cross-section images are shown in Figure 4. The results evidenced full coverage of the samples’ surface, but with significant differences in the morphology depending on the coatings’ architecture. All the coatings revealed a large distribution of grain sizes (Figure 4a–c), except for the Zn-Fe alloy, which Nanomaterials 2022,12, 2104 7 of 14 showed a more compact morphology (Figure 4b) than all the other samples. Both the Zn NLs + Fe NPs and Zn-Fe NLs had heterogeneous particle distributions, but the latter showed a clear bimodal particle size distribution. Nanomaterials 2022, 12, x FOR PEER REVIEW 8 of 15 Figure 4. SEM surface images of the coatings: (a) pure Zn; (b) Zn-Fe alloy; (c) Zn NLs + Fe NPs; and (d) Zn-Fe NLs. The inset in each image represents the SEM cross-section for each sample. Table 2 shows that the Zn at.% was approximated (81–86 at.%) for most of the samples, except for the Zn-Fe alloy coating, which featured almost half of this composition (46 at.%). The Zn-Fe NL and Zn NL + Fe NP nanostructures presented very close Fe at.% values, while Zn-Fe alloy, as expected, had a higher amount of Fe. The O at.% was lowest for the Zn-Fe NLs (4 at.%), similar for the pure Zn and Zn NLs + Fe NPs (14 and 11 at.%, respectively), and around 30 at.% for the Zn-Fe alloy. It is worth noting that in a previous study, Zn-Fe alloys with 9 at.% of Fe showed passivation against the oxidation of the coating [23]. Thus, in the present investigation, the Zn-Fe alloy sample was produced with a higher amount of Fe to promote the oxidation of the film for the expected chromatic and antibacterial properties. The presence of oxygen in all the samples was ascribed to a spontaneous oxidation reaction when the small metal nanostructures were in contact with air. Table 2. Experimental details used during deposition, thickness, atomic composition, and final mass concentration of Zn and Fe in the produced nanostructures. Deposit Conditions Characteristics of the Produced Coatings Coatings Deposition time (min) Current Density (J) (mA/cm 2 ) Deposition Rate (nm/s) Thickness (nm) Atomic Percent (at.%) Metal Concentration by ICPOES (µg/cm 2 ) J zn J Fe Zn Fe O Zn Fe Pure Zn Zn = 14.5 0.5 — 0.13 109 86 — 14 27.40 ± 0.43 — Zn-Fe alloy Zn = 15 Fe = 62.5 0.5 2.5 0.30 175 46 24 30 33.90 ± 0.60 13.51 ± 0.97 Zn NLs + Fe NPs Zn = 12 Fe = 240 2.5 3.2 0.02 238 81 8 11 47.13 ± 4.21 1.40 ± 0.52 Zn-Fe NLs Zn = 15 Fe = 25 1* 2.5* 0.09 207 89 7 4 69.20 ± 1.34 3.77 ± 0.11 * Zn and Fe simultaneously deposited. Figure 4. SEM surface images of the coatings: ( a ) pure Zn; ( b ) Zn-Fe alloy; ( c ) Zn NLs + Fe NPs; and (d) Zn-Fe NLs. The inset in each image represents the SEM cross-section for each sample. Table 2shows that the Zn at.% was approximated (81–86 at.%) for most of the samples, except for the Zn-Fe alloy coating, which featured almost half of this composition (46 at.%). The Zn-Fe NL and Zn NL + Fe NP nanostructures presented very close Fe at.% values, while Zn-Fe alloy, as expected, had a higher amount of Fe. The O at.% was lowest for the Zn-Fe NLs (4 at.%), similar for the pure Zn and Zn NLs + Fe NPs (14 and 11 at.%, respectively), and around 30 at.% for the Zn-Fe alloy. It is worth noting that in a previous study, Zn-Fe alloys with 9 at.% of Fe showed passivation against the oxidation of the coating [ 23 ]. Thus, in the present investigation, the Zn-Fe alloy sample was produced with a higher amount of Fe to promote the oxidation of the film for the expected chromatic and antibacterial properties. The presence of oxygen in all the samples was ascribed to a spontaneous oxidation reaction when the small metal nanostructures were in contact with air. The total concentration of Zn and Fe of the samples is also shown in Table 2. The sample with the highest Zn concentration was the Zn-Fe NLs, followed by the Zn NL + Fe NP, Zn-Fe alloy, and pure Zn coatings. Although the thicknesses of the Zn-Fe NL and Zn NL + Fe NP coatings were similar, the different production methods led to modifications in the morphologies and therefore in the concentrations. XPS depth profiles were carried out to study the chemical bonding of the coatings as a function of the thickness for Fe 2p, O 1s, and Zn LMM. It is important to highlight that the X-ray-induced Zn LMM Auger peaks had a larger shift with the chemical state than those of metallic Zn and ZnO, and therefore, this was preferred over Zn 2p. Figure 5shows the evolution of the Zn, Fe, and O as a function of the argon sputtering time, showing that the coatings were composed of a metallic and oxidized mixture of Zn and Fe. Nanomaterials 2022,12, 2104 8 of 14 Table 2. Experimental details used during deposition, thickness, atomic composition, and final mass concentration of Zn and Fe in the produced nanostructures. Deposit Conditions Characteristics of the Produced Coatings Coatings Deposition time (min) Current Density (J) (mA/cm2)Deposition Rate (nm/s) Thickness (nm) Atomic Percent (at.%) Metal Concentration by ICP-OES (µg/cm2) Jzn JFe Zn Fe O Zn Fe Pure Zn Zn = 14.5 0.5 — 0.13 109 86 — 14 27.40 ±0.43 — Zn-Fe alloy Zn = 15 Fe = 62.5 0.5 2.5 0.30 175 46 24 30 33.90 ±0.60 13.51 ±0.97 Zn NLs + Fe NPs Zn = 12 Fe = 240 2.5 3.2 0.02 238 81 8 11 47.13 ±4.21 1.40 ±0.52 Zn-Fe NLs Zn = 15 Fe = 25 1 * 2.5 * 0.09 207 89 7 4 69.20 ±1.34 3.77 ±0.11 * Zn and Fe simultaneously deposited. Nanomaterials 2022, 12, x FOR PEER REVIEW 9 of 15 The total concentration of Zn and Fe of the samples is also shown in Table 2. The sample with the highest Zn concentration was the Zn-Fe NLs, followed by the Zn NL + Fe NP, Zn-Fe alloy, and pure Zn coatings. Although the thicknesses of the Zn-Fe NL and Zn NL + Fe NP coatings were similar, the different production methods led to modifications in the morphologies and therefore in the concentrations. XPS depth profiles were carried out to study the chemical bonding of the coatings as a function of the thickness for Fe 2p, O 1s, and Zn LMM. It is important to highlight that the X-ray-induced Zn LMM Auger peaks had a larger shift with the chemical state than those of metallic Zn and ZnO, and therefore, this was preferred over Zn 2p. Figure 5 shows the evolution of the Zn, Fe, and O as a function of the argon sputtering time, showing that the coatings were composed of a metallic and oxidized mixture of Zn and Fe. Figure 5. XPS spectra of the coating as a function of sputtering time: (a) pure Zn; (b) Zn-Fe alloy; (c) Zn NLs + Fe NPs; and (d) Zn-Fe NLs. Only a fraction of the Zn and Fe was oxidized, which accounted for the passivation of the metals at the surface already observed in the STEM images. The pure Zn sample (Figure 5a) also showed a combination of Zn/ZnO, which was attributed to the passivation of Zn in a natural environment. The incorporation of Fe in the coatings intensified the oxidation of Zn, as evidenced by the intensity ratio between the ZnO peak located at 988 eV [34] and the metallic Zn peak at 992 eV [35], as shown in Figure 5b–d. This effect was likely potentiated by the galvanic couple created by the Zn and Fe. The oxidation of Zn and Fe was much more pronounced in the Zn-Fe alloy coating (Figure 5b). Furthermore, the three samples with Zn and Fe (Figure 5b–d) contained Fe in both metallic oxidized states. The components located at 706 and 720 eV were attributed to Fe-Fe, whereas those at 711 and 724 eV were attributed to Fe3+ in Fe2O3 [36,37]. Note that no significant shifts in the peak location were observed as a function of thickness or etching time. All samples revealed two peaks in the O 1s, with different intensity ratios. The first peak was located at 530.7 eV, related to the O atoms in the metal oxides. The second, at 532 eV, was attributed to O in oxygen-deficient regions within the matrix of ZnO, which explains the changes in the intensity of this peak for all the samples, since variation in the concentration of oxygen vacancies were expected [38]. The XPS O 1s patterns of the Zn NL + Fe NP coating (Figure 5c) were most intense, which was due to the high amount of O that was already noticed with the at.% of oxygen in Table 2. Furthermore, the presence Figure 5. XPS spectra of the coating as a function of sputtering time: ( a ) pure Zn; ( b ) Zn-Fe alloy; (c) Zn NLs + Fe NPs; and (d) Zn-Fe NLs. Only a fraction of the Zn and Fe was oxidized, which accounted for the passivation of the metals at the surface already observed in the STEM images. The pure Zn sample (Figure 5a) also showed a combination of Zn/ZnO, which was attributed to the passivation of Zn in a natural environment. The incorporation of Fe in the coatings intensified the oxidation of Zn, as evidenced by the intensity ratio between the ZnO peak located at 988 eV [ 34 ] and the metallic Zn peak at 992 eV [ 35 ], as shown in Figure 5b–d. This effect was likely potentiated by the galvanic couple created by the Zn and Fe. The oxidation of Zn and Fe was much more pronounced in the Zn-Fe alloy coating (Figure 5b). Furthermore, the three samples with Zn and Fe (Figure 5b–d) contained Fe in both metallic oxidized states. The components located at 706 and 720 eV were attributed to Fe-Fe, whereas those at 711 and 724 eV were attributed to Fe 3+ in Fe 2 O 3 [ 36 , 37 ]. Note that no significant shifts in the peak location were observed as a function of thickness or etching time. All samples revealed two peaks in the O 1s, with different intensity ratios. The first peak was located at 530.7 eV, related to the O atoms in the metal oxides. The second, at 532 eV, was attributed to O in oxygen-deficient regions within the matrix of ZnO, which explains the changes in the intensity of this peak for all the samples, since variation in the concentration of oxygen vacancies were expected [ 38 ]. The XPS O 1s patterns of the Zn Nanomaterials 2022,12, 2104 9 of 14 NL + Fe NP coating (Figure 5c) were most intense, which was due to the high amount of O that was already noticed with the at.% of oxygen in Table 2. Furthermore, the presence of the oxygen peak at higher energies for the final etching time corresponded to the C-O and C=O for PLA. As shown in Figure 6, selected area electron diffraction was acquired from samples observed from the top view, showing the presence of polycrystalline materials for pure Zn (Figure 6a), Zn NL + Fe NP (Figure 6c), and Zn-Fe NL (Figure 6d) coatings. However, the Zn-Fe alloy (Figure 6b) exhibited quasiamorphous phases but with distinctive rings. The pattern profiles of the samples are overlaid in Figure 6. The peaks of pure Zn matched well with a mixture of Zn and ZnO phases, both hexagonal. More diffused rings were observed in the coatings with the incorporation of Fe, indicating smaller crystals in the Zn-Fe coatings due to the Fe addition. No evidence of additional phases was observed in the Zn-Fe samples, despite the clear existence of isolated Fe layers observed in the cross-section images. In the Zn-Fe alloy coating, two large peaks were observed, which are indistinguishable from the Zn, ZnO, or Fe2O3phases. Nanomaterials 2022, 12, x FOR PEER REVIEW 10 of 15 of the oxygen peak at higher energies for the final etching time corresponded to the C-O and C=O for PLA. As shown in Figure 6, selected area electron diffraction was acquired from samples observed from the top view, showing the presence of polycrystalline materials for pure Zn (Figure 6a), Zn NL + Fe NP (Figure 6c), and Zn-Fe NL (Figure 6d) coatings. However, the Zn-Fe alloy (Figure 6b) exhibited quasiamorphous phases but with distinctive rings. The pattern profiles of the samples are overlaid in Figure 6. The peaks of pure Zn matched well with a mixture of Zn and ZnO phases, both hexagonal. More diffused rings were observed in the coatings with the incorporation of Fe, indicating smaller crystals in the Zn-Fe coatings due to the Fe addition. No evidence of additional phases was observed in the Zn-Fe samples, despite the clear existence of isolated Fe layers observed in the crosssection images. In the Zn-Fe alloy coating, two large peaks were observed, which are indistinguishable from the Zn, ZnO, or Fe 2 O 3 phases. Figure 6. SAED images obtained for each coating: (a) pure Zn; (b) Zn-Fe alloy; (c) Zn NLs + Fe NPs; and (d) Zn-Fe NLs. 3.2. Functional Properties 3.2.1. Chromatic Properties Figure 7a, b shows the color and opacity of the coatings, respectively, before and after exposing them to a high relative humidity (RH) environment for 7 days (RH = 98%) with the aim to promote the oxidation of the metallic nanostructures. Figure 6. SAED images obtained for each coating: ( a ) pure Zn; ( b ) Zn-Fe alloy; ( c ) Zn NLs + Fe NPs; and (d) Zn-Fe NLs. 3.2. Functional Properties 3.2.1. Chromatic Properties Figure 7a, b shows the color and opacity of the coatings, respectively, before and after exposing them to a high relative humidity (RH) environment for 7 days (RH = 98%) with the aim to promote the oxidation of the metallic nanostructures.