2.1. Laser Ablation
A Copper foil with 99.99% of purity (Alfa Aesar) previously cleaned, was used as a laser ablation target. Titanium discs Grade 2 (Goodfellow Cambridge Limited, Huntingdon, UK) with 10 mm diameter and about 200 nm of average surface roughness were used as substrates to collect the ablated material. The target was set at a 30 degree angle with the horizontal plane, and the laser beam was focused on its upper surface. In each experiment, one Ti substrate was tilted to be placed almost parallel to the copper target, 10 mm away from the incident point of the laser beam, as detailed in
Figure 1.
Two different laser sources were used in the process. The first system was a diode-pumped Nd:YVO4 laser, providing pulses of 14 ns at a wavelength of 532 nm with 0.26 mJ of pulse energy. The second laser source was a Nd:YVO4 laser providing pulses of 800 ps at 1064 nm of wavelength and 0.03 mJ of pulse energy. The laser beam spot diameter on the target surface was estimated to be 132 μm, giving a fluence of 1.90 J/cm
2 in the case of the Green-Nanosecond laser and 196 μm, giving a fluence of 0.09 J/cm
2, for the IR–Picosecond laser. In all cases, the laser beam was kept in relative movement with respect to the target at 50 mm/s of scanning speed. The processing parameters used are listed in
Table 1.
To analyze in depth the formation process of nanoparticles and the influence of oxygen on this process, two different atmospheres were used with each laser source: In open air and in the argon environment by using an airtight chamber which kept the oxygen content below 50 ppm during the process. Note that all the assays were performed at atmospheric pressure (1 atm). Sample nomenclature with the corresponding assay conditions are listed in
Table 2.
In order to compare the obtained samples, the same ablated mass (2 mg) for each condition was deposited on a titanium plate. Since the process parameters (shown in
Table 1) and atmosphere changed, different processing time was required to obtain the same ablated mass as reported in
Table 2. In this sense, it is noteworthy to mention that several previous assays were performed with each condition before the final preparation, in order to adjust carefully the required time to obtain the same mass. Additionally, in each final assay, the ablated mass was confirmed by weighing the targets before and after the ablation process (
± 0.001 g) for a higher accuracy.
2.2. Sample Preparation and Characterization Technics
The morphology and composition of the nanoparticles on the titanium surface were characterized by using a Ga ion beam in the FEI Helios NanoLab 600 (FEI, Hillsboro, OR, USA) dual beam microscope. Scanning electron microscopy (SEM) micrographs and energy dispersive spectroscopy X-rays (EDS) on the sectioning layer of the coating were obtained by Focused Ion Beam (FIB).
The deposited nanoparticles on the titanium discs were observed by Field Emission Scanning Electron Microscopy (FESEM) with a JEOL JSM 6700F microscope (JEOL, Akishima, Japan). Carbon-coated copper microgrids were also used as substrates to collect the nanoparticles for a size and morphology analysis. Transmission Electron Microscopy (TEM) images were acquired with a JEOL JEM 1010 (JEOL, Akishima, Japan) microscope and the nanoparticle size distribution was derived from a histogram obtained by measuring the diameter of about 300 particles.
Nanoparticles were also deposited on carbon-free copper microgrids in order to accomplish a crystallographic characterization. High-Resolution Transmission Electron Microscopy (HRTEM) and Selected Area Electron Diffraction (SAED) images were acquired with a JEOL JEM 2010F (JEOL, Akishima, Japan) high-resolution transmission electron microscope, equipped with a slow digital camera scan, using a 200 kV accelerating voltage. Identification of phases was achieved by comparing the measured distances with the diffraction patterns from the ICDD (JCPDS) database.
In order to corroborate the composition of the obtained nanoparticles, X-ray diffraction (XRD) analysis was carried out by means of a PANanalytical X’Pert Pro X-ray diffractometer using monochromated Cu-Kα radiation (wavelength 1.54 Å) over the 20–100° 2θ range with a step size of 0.02°. To facilitate the phase identification, the nanoparticles were deposited on a zero-background holder. The diffraction peaks of each sample were compared with the reference pattern of pure copper and different copper oxides from the ICDD (JCPDS) database.
The same ablation process was repeated by using a piece of glass to collect the nanoparticles, with the purpose of studying the optical properties. The ultraviolet to visible (UV/VIS) absorption spectrum of the copper nanoparticles’ layer was measured in the range from 280 to 800 nm, using a Hewlett Packard HP 8452 diode array spectrophotometer.
Finally, three replicas (titanium discs with NPs) of each condition were submerged in 25 mL of ultrapure, deionized water in order to study the influence of copper ions in the bactericidal process. From each replica, 1.5 mL were laid away regularly during the first 21 days to be centrifuged. The extracted volume (1.5 mL) was gently replaced with fresh, deionized water. Afterwards, possible nanoparticles were separated from the solutions by using an Eppendorf miniSpin centrifuge at 13,400 rpm for 30 min at room temperature. After centrifuging, only 1 mL from the upper surface was taken, leaving the heaviest matter (NPs) in the bottom. In this regard, in order to ensure that only the ions in suspension are measured, UV-VIS spectroscopy was also used before analyzing. Finally, the ions content in the solutions was measured by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) with an Optima 4300 DV (Perkin Elmer, Waltham, MA, USA).
2.3. Antimicrobial Activity
The antibacterial activity of the treated samples was studied with Staphylococcus aureus (CECT (Colección Española de Cultivos Tipo) 435, Valencia, Spain) cultured in BHI broth (Scharlab SL, Sentmenat, Spain). A bacteria inoculum was incubated for 24 h at 37 °C before the assay.
The optical density for the bacterial suspension was adjusted to 0.2 ± 0.01 at 600 nm, equivalent to 1 × 108 colony-forming units (CFU)/mL.
The control and treated samples were immersed in ethanol and distilled water for 15 min each, and put into a 24-multiwell plate (Nunc, Rochester, NY, USA) with 1 mL bacterial suspension at 37 °C for 2 h. Afterwards, the samples were washed thrice with phosphate buffered solution (PBS) to wash off the nonadherent bacteria. The adhered bacteria were collected, sonicating the samples in 1 mL sterile PBS for 5 min. The PBS was serially diluted, and the diluted bacterial suspensions were seeded onto agar plates supplemented with BHI medium. The agar plates were then incubated at 37 °C for 24 h, and the CFUs were counted.