[Al2o3/Zno]N/316L SS Nanolaminates Growth by Atomic Layer Deposition (ALD)

crystals

Article Structure and Surface Morphology Eﬀect on the Cytotoxicity of [Al2O3/ZnO]n/316L SS Nanolaminates Growth by Atomic Layer Deposition (ALD)

D. Osorio 1, J. Lopez 2 , H. Tiznado 3 , Mario H. Farias 3, M. A. Hernandez-Landaverde 4, M. Ramirez-Cardona 5 , J. M. Yañez-Limon 4, J. O. Gutierrez 6, J. C. Caicedo 7 and G. Zambrano 1,*

1 Thin Films Group, Universidad del Valle, Cali C.P. 76001, Colombia; [email protected] 2 CONACYT—Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, Ensenada, B.C, Av. Insurgentes Sur 1582, Ciudad de Mexico C.P. 03940, Mexico; [email protected] 3 Centro de Nanociencias y Nanotecnología (CNyN), Universidad Nacional Autónoma de México (UNAM), Km 107 Carretera Tijuana-Ensenada s/n. B. C., Mexico C.P. 22800, Mexico; [email protected] (H.T.); [email protected] (M.H.F.) 4 Centro de Investigación y de Estudios Avanzados del I.P.N., Unidad Querétaro, Libramiento Norponiente No. 2000, Fracc. Real de Juriquilla, Querétaro Qro. C.P. 76230, Mexico; [email protected] (M.A.H.-L.); [email protected] (J.M.Y.-L.) 5 Área Académica de Ciencias de la Tierra y Materiales, Universidad Autónoma del Estado de Hidalgo (UAEH), Ciudad del Conocimiento, Col. Carboneras, Mineral de la Reforma Hgo. C.P. 42184, Mexico; [email protected] 6 Farmacología Univalle, Universidad del Valle, Cali C.P. 76001, Colombia; [email protected] 7 TPMR Group, Universidad del Valle, Cali C C.P. 76001, Colombia; [email protected] * Correspondence: [email protected]

Received: 12 June 2020; Accepted: 3 July 2020; Published: 16 July 2020

Abstract: Recently, different biomedical applications of aluminum oxide (Al2O3) and zinc oxide (ZnO) have been studied, and they have displayed good biocompatible behavior. For this reason, this study explores nanolaminates of [Al2O3/ZnO]n obtained by atomic layer deposition (ALD) on silicon (100) and 316L stainless steel substrates with different bilayer periods: n = 1, 2, 5, and 10. The intention is to correlate the structure, chemical bonds, morphology, and electrochemical properties of ZnO and Al2O3 single layers and [Al2O3/ZnO]n nanolaminates with their cytotoxic and biocompatibility behavior, to establish their viability for biomedical applications in implants based on the 316L SS substrate. These nanolaminates have been characterized by grazing incident X-ray diffraction (XRD), finding diffraction planes for wurtzite type structure from zincite. The chemical bonding and composition for both single layers were identified through X-ray photoelectron spectroscopy (XPS). The morphology and roughness were tested with atomic force microscopy (AFM), which showed a reduction in roughness and grain size with a bilayer period increase. The thickness of the samples was measured with scanning electron microscopy, and the results confirmed the value of ~210 nm for the nanolaminate samples. The electrochemical impedance spectroscopy analysis with Hank’s balanced salt solution (HBSS) evidenced an evolution of [Al2O3/ZnO]n/316L system corrosion resistance of around 95% in relation with the uncoated steel substrate as function of the increase in the bilayers number. To identify the biocompatibility behavior of these nanolaminate systems, the lactate dehydrogenase test was performed with Chinese hamster ovary (CHO) cells for a short system of life cell evaluation. This test shows the cytotoxicity of the multilayer compared to the single layers of Al2O3, ZnO, and 316L stainless steel. The lowest cytotoxicity was found in the single layers of ZnO, which leads to cell proliferation easier than Al2O3, obtaining better adhesion and anchoring to its surface.

Crystals 2020, 10, 620; doi:10.3390/cryst10070620 www.mdpi.com/journal/crystals Crystals 2020, 10, 620 2 of 16

Keywords: nanolaminates; Al2O3; ZnO; X-ray diﬀraction; lactate dehydrogenase; cytotoxicity

1. Introduction Multilayer systems show the best interaction with different materials of natural origin for a specific use. An example of this is the nanolaminates of different thin film oxides. These forms have many utilities and have been studied for their magnetic, electrical, optical, mechanical, electrochemical, and biocompatible properties. However, to explore the potential applications of nanolaminates it is relevant that nanostructures have novel properties compared to thicker films or bulk material, especially given that the conjugation of the number of interfaces and the nature of the multilayer system happens in the same way as the superlattice systems [1]. The consistency of the atomic layer deposition (ALD) films lies in the gas phase thin film deposition method, as a modification of chemical vapor deposition (CVD). For the ALD process, the precursor material is led into the chamber by alternating cycles or pulses, where any cycles are separated by an inert gas purging or by evacuation in high-vacuum reactors [1]. The precursors react on the substrate surface according to the process conditions, and when some precursors are self-limited it leads to a controlled film growth by surface saturated reactions. An example of these is the nanolaminates of aluminum oxide (Al2O3)/zinc oxide (ZnO) deposited by ALD [2–8], which has improved the optical or mechanical behavior of the single layer with the bilayers number increase [9–11]. Both materials come from self-limiting organometallic precursors such as trimethyl-aluminum (TMA) and diethylzinc (DEZ), and both represent a scheme for other applications using the individual potential of single Al2O3 and ZnO layers. For the electro-catalytic process, both oxides have been studied with glucose oxidation [12]; even the mechanical properties of these oxides are important to notice because their multilayer structure leads to an increase in hardness and toughness with the bilayer period according to the Hall-Petch effect [9]. Biocompatible behavior is an important property for these materials too; ZnO has been studied to check its biocompatibility and biosafety, and in some cases, it is considered to be a nanosensor for implantable biomedical detections [13]. Some authors have shown their cell viability for many potential applications [8,14–18]. Similarly, Al2O3 exhibits a biocompatible behavior and is a standard ceramic–ceramic articulation material, due to its hardness and its least wear compared to metals, it has been used as the femoral head for joint replacement through decades [13,19]. Given that both ZnO and Al2O3 show biocompatible behavior, it would be interesting to show how these materials can be approached and what kind of interactions can be obtained for the nanolaminates of both materials. Especially, to improve the mechanical behavior, wear rate or biocompatible behavior in multilayer system, as it is the objective of this study. This study used the atomic layer deposition (ALD) technique. Through this process, aluminum oxide (Al2O3) and zinc oxide (ZnO) was deposited in the form of nanostructured layers with the same ratio between the single layers and different bilayer periods (λ), for the same final thickness of the Al2O3/ZnO bilayers. The ALD technique has emerged as a process to obtain thin films in the nanoscale regimen [20,21] with the advance in semiconductors, especially for oxides in metal oxide semiconductor field effect Transistor (MOSFET) structures, capacitors for dynamic random access memory (DRAM), integrated circuits and other microelectronic devices [20,22]. Many studies on ALD report that it can obtain novel materials grown with good thickness uniformity and conformality into three-dimensional structures, however, some of its potential applications could be for protective coatings, as anti-corrosive films for biocompatible films [23,24] In this work, we want to study the effect on the structure, chemical bonds, morphology, and electrochemical properties of ZnO and Al2O3 single layers and [Al2O3/ZnO]n multilayer nanolaminates system deposited on 316L SS substrate. The above will correlate with its cytotoxic and biocompatibility behavior to establish its viability for biomedical applications in implants based on the 316L SS substrate. Crystals 2020, 10, x 3 of 16

will correlate with its cytotoxic and biocompatibility behavior to establish its viability for biomedical applications in implants based on the 316L SS substrate.

Crystals 2020, 10, 620 3 of 16 2. Experimental Details

Multilayers or Al2O3/ZnO nanolaminates were deposited by the atomic layer deposition 2. Experimental Details technique, with a Beneq (Espoo, Uusimaa, Finland) TFS 200 ALD reactor using gas precursors of Multilayersthrymetilaluminum or Al2O (TMA,3/ZnO Strem nanolaminates 93-1360) and were diethylzinc deposited (DEZ, by Aldrich the atomic 256781) layer, so that deposition during each technique,cycle, thicknesses with a Beneq of 1.2 (Espoo, Å and Uusimaa, 2 Å can be Finland) obtained TFS for the 200 Al ALD2O3 and reactor ZnO using layers, gas respectively. precursors Ultra of thrymetilaluminumhigh purity (UHP) (TMA, nitrogen Strem (99.999%) 93-1360) wa ands introduced diethylzinc to the (DEZ, chamber Aldrich as 256781),a carrier/purge so that duringgas with 1 × −12 each10 cycle, ppm thicknesses and a flow of 1.2rate Å of and 250 2sccm. Å can For be all obtained of the processes for the Al a2 Oconstant3 and ZnO substrate layers, temperature respectively. of 200 Ultra°C high and purity a ~ 1.5 (UHP) mbar nitrogen reactor pressure (99.999%) were was used. introduced Vapor to deionized the chamber water as awas carrier the /oxidantpurge gas agent. with 1 10 12 ppmSi (100) and and a flow 316L rate stainless of 250 sccm. steel For used all as of thesubstrates processes were a constant previously substrate adapted temperature and cleaned of to × − 200 ◦Cprevent and a ~1.5 any mbar conta reactormination pressure on the were surface. used. To Vapor achieve deionized this, mechanical water was the polishing oxidant andagent. chemical Sicleaning (100) and were 316L used. stainless First, the steel 316L used stainless as substrates steel (316L were SS) was previously mechanically adapted machined and cleaned to obtain to flat preventdiscs any of contamination1mm thickness on and the 1cm surface. of diameter. To achieve Later, this, these mechanical steel substrates polishing, together and chemical with those of cleaningsilicon were, were used. cleaned First, thein acetone 316L stainless with ultrasound steel (316L for SS) 15 was min mechanically at 50°C, then machined rinsed with to obtainan isopropanol flat discsreagent of 1 mm grade thickness, and finally and 1 dried cm of with diameter. UHP N Later,2. To theseobtain steel the Al substrates,2O3 layer, togethercycles of with150 ms those TMA of dose, silicon,750 were ms N cleaned2 purge, in and acetone 150 ms with DI ultrasound water dose for were 15 minintroduced at 50◦C, into then the rinsed chamber with, anand isopropanol then 500 ms N2 reagentpurge grade, for andthe vacuum finally dried process. with The UHP chemical N2. To reactions obtain the with Al2O the3 layer, TMA cycles follow of the 150 Equation ms TMA (1 dose,) [25,26] . 750 msAfterward, N2 purge, UHP and N 1502 purge ms DI the water chamber dose before were introduced starting another into the cycle. chamber, Similarly, and to then obtain 500 ZnO ms N layers,2 purge30 for ms the DEZ vacuum dose, process.2 s N2 purge, The chemical150 ms DI reactions water dose, with and the TMA500 ms follow N2 purge, the Equation are needed. (1) [These25,26]. react Afterward,through UHP Equation N2 purge (2) [27 the– chamber29] to obtain before an startingatomic layer another per cycle.cycle. Similarly, to obtain ZnO layers, 30 ms DEZ dose, 2 s N2 purge, 150 ms DI water dose, and 500 ms N2 purge, are needed. These react 2() + 3 → + 3 (1) through Equation (2) [27–29] to obtain an atomic layer per cycle.

2Al(CH3)3 + 3H2O Al2O3 + 3CH4 (1) () + () (→) → − () + () (2) (OH()− +) Zn−( CH()()g )+ O (Zn) →(CH )−+(C H) +(g ) () 2 52 → − − 2 5 2 6 (2) (OH)− Zn(C H ) + H O (g) OZn (OH) + C H (g) − 2 5 2 → − 6 6 The processesThe processes described described wererepeated were repeated in many in cycles, many ascycles, the thickness as the thickness needs to completeneeds to complete the ﬁlm. the Thesefilm. [Al2 O These3/ZnO] [Aln 2nanolaminatesO3/ZnO]n nanolaminates were deposited were with deposited a thickness with ratio a thickness of 1:1, for ratio a total of 1:1, thickness for a total set constantthickness with set a constant nominal valuewith a of nominal ~200 nm value and di ofﬀ erent~200 bilayersnm and perioddifferentn (1, bilayers 2, 5, and period 10). The n (1, above 2, 5, and means10). that The the above thickness means of that each the ZnO thickness and Alof3 eachO3 layer ZnO wasand 100,Al3O 50,3 layer 20, w oras 10 100, nm 50, for 20n, or= 1,10 2,nm 5, for n and 10= 1, bilayer 2, 5, and periods 10 bilayer, respectively. periods, Figurerespecti1 showsvely. Figure a scheme 1 shows of the a [Al scheme2O3/ZnO] of then multilayer[Al2O3/ZnO] structuren multilayer obtainedstructure during obtained the ALD during process. the ALD process.

Figure 1. Experimental configurations for the [aluminum oxide (Al2O3)/zinc oxide (ZnO)] n multilayer structure. Figure 1. Experimental configurations for the [aluminum oxide (Al2O3)/zinc oxide (ZnO)]n multilayer structure. Different methods were used to characterize the nanolaminate systems of Al2O3 and ZnO single layers, deposited onto silicon and 316 SS substrates. Phase identification of ALD deposited layers on silicon substrate (100) was accomplished by patterns obtained from grazing incidence X-ray diffraction (GIXRD) experiments (Ultima IV diffractometer, RIGAKU, Akishima-shi, Tokyo, Japan). Crystals 2020, 10, 620 4 of 16

The experiments were conducted on a RIGAKU Ultima IV diffractometer, using CuKα1,2 radiation (1.5406 Å/1.5444 Å doublet wavelength generated by an X-ray tube operating at 40 kV and 30 mA), in a 2θ range from 22◦ to 72◦, a step scan of 0.02◦ and an integration time of 0.5 s per step. The parallel incident beam was fixed at an angle of 1◦ with the sample surface. To determine the chemical composition, X-ray photoelectron spectroscopy was employed using a JEOL JPS 9200 spectrometer (Tokyo, Japan) with monochromatic Al Kα line. The roughness and surface morphology were studied using atomic force microscopy (AFM) (XE 70, Santa Clara, CA, USA). The SEM instrument was a JEOL JIB-4500 (Hesperia, CA, USA) Focused Ion Beam Scanning Electron Microscope and the Field Emission Electron TEM microscope (JEOL, Peabody, MA, USA) was a JEOL JEM-2100F. For TEM analysis, the witness sample growth on Si(100) substrate was examined. Additionally, the electrochemical behavior of [Al2O3/ZnO]n/316L SS system was studied by the electrochemical impedance spectroscopy (EIS) method in the presence of a Hank’s balanced salt solution (HBSS). Finally, the cytotoxicity test lactate dehydrogenase (LDH) used gave us a first summary of the biocompatibility for these materials. The electrochemical study was carried out with a Gamry unit, model PCI 4, utilized for DC and AC measurements. Electrochemical impedance spectroscopy (EIS) and Tafel polarization curves were obtained at room temperature, using a cell with a working electrode within an exposed area of 1 cm2, a reference electrode (Ag/AgCl), and a platinum wire counter-electrode under Hank´s balanced salt solution (HBSS) for an exposure time of 30 min. For Nyquist diagrams, the frequency sweep was performed in the range from 100 kHz to 0.001 Hz using sinusoidal voltage amplitude of 10 mV applied to the working electrode (sample) and reference electrode. To obtain Tafel polarization curve diagrams a voltage sweep was carried out at a speed of 0.5 mV/s in the range of 0.25 to 0.25 V. To determine the roughness and grain size of the top layer, an MFP-3D Asylum − Research atomic force microscope (AFM) was used. A cytotoxicity test provides information about direct cell interaction with a chemical compound, pharmaceutics, or biomaterials. There are many methods in the literature related to cytotoxicity tests. A cytotoxicity test was carried out with a lactate dehydrogenase (LDH) kit plus version 0.6, Roche, Mannhelm, Germany [30], which provides a simple, reliable colorimetric method for quantifying cellular cytotoxicity by the liberation of lactate dehydrogenase (plasma membrane damage releases LDH into the cell culture media [31] Chinese hamster ovary (CHO) cell cultures were done to obtain 50,000 cells/well, for use in triplicate samples. For the cytotoxicity test, a 24-well plate with different distributions for the sample was available, as well as for the positive (releasable triton) and negative controls in this LDH assay. These cell cultures were deposited on the surface of ZnO, Al2O3, [Al2O3/ZnO]n/316L SS substrate, and copper was used as a positive cytotoxic control. After 24 h the cytotoxic LDH was applied to the culture, and the reaction mixture gets incubated for 30 min until a stop solution was applied. The enzymatic assay was performed in the supernatant and it was measured in an Elisa spectrometer, with an absorbance reading from 450 to 630 nm. The absorbance readings for each the samples were compared to a control reagent for positive and negative cytotoxicity, for this LDH kit the Equation (3) was used [30].

Exp.Value low control Cytotoxicity (%) = − 100 (3) high control low control × − Finally, for the cell adhesion analysis to the surface of the samples after spending 48 h in contact with them, a cell ﬁxation process was used employing histological techniques using a mixture of 3% glutaraldehyde (GA) in phosphate-buﬀered solution (PBS) to maintain the osmolarity and pH of the aqueous solution of the cells. After this process, the surface of the samples was analyzed under the scanning electron microscope. Crystals 2020, 10, 620 5 of 16 Crystals 2020, 10, x 5 of 16

3. Results 3.1. X-ray Diffraction Analysis 3.1. X-rayFor comparative Diffraction Analysis purposes, a GIXRD study of nanolaminates growth on Si (100) wafer, and 316L SS substrateFor comparative is carried purposes, out. Figure a GIXRD 2 shows study the ofdiffraction nanolaminates patterns growth of Al on2O3 Siand (100) ZnO wafer, single and layers, 316L and [Al2O3/ZnO]n nanolaminates with n = 1, 2, 5 and 10, for both 316L SS (Figure 2a) and Si(100) SS substrate is carried out. Figure2 shows the di ffraction patterns of Al2O3 and ZnO single layers, (Figure 2b) wafer substrates. The corresponding GIXRD single-layer patterns corroborated the and [Al2O3/ZnO]n nanolaminates with n = 1, 2, 5 and 10, for both 316L SS (Figure2a) and Si(100) (Figureexpected2b) amorphous wafer substrates. nature The of correspondingAl2O3. ZnO, for GIXRD both a single-layer single layer patterns or in multilayers, corroborated crystallizes the expected on wurtzite-type structure (zincite, space group P63mc, a = b = 3.24982 Å, c = 5.20661 Å, α = β = 90°, γ = amorphous nature of Al2O3. ZnO, for both a single layer or in multilayers, crystallizes on wurtzite-type 120°, from JCPDS card 36-1451 [32]). In the case of the steel-substrate, its patterns clearly exemplified structure (zincite, space group P63mc, a = b = 3.24982 Å, c = 5.20661 Å, α = β = 90◦, γ = 120◦, from JCPDS cardthe main 36-1451 peaks [32 of]). α In- and the caseγ-Fe offorms. the steel-substrate, For samples growth its patterns onto the clearly Si wafer, exemplified its (100) thepeak main was peaks slightly of αdetected,- and γ-Fe and forms. the Forspecific samples combination growth onto between the Si wafer, the incident its (100) grazing peak was angle slightly (1°) detected, and the and sample the orientation in the holder produced the low intensity of that peak. Patterns from the ZnO single layer specific combination between the incident grazing angle (1◦) and the sample orientation in the holder produceddeposited thedirectly low intensity on the substrate of that peak. show Patternsed multiaxial from thepreferred ZnO single orientation layer deposited models: (100)/(002) directly on and the substrate(100)/(002)/(103) showed formultiaxial 316L SS preferred and Si orientation substrates, models: respectively; (100)/(002) and and only (100) the/(002) principal/(103) for preferred 316L SS andorien Sitation substrates,—i.e., respectively; PO on (002) and plane only the of principal zincite—wa preferreds maintained orientation—i.e., through PO the on [Al (002)2O3/ZnO] planen nanolaminate samples. The former two PO’s models are the result of the different substrates’ of zincite—was maintained through the [Al2O3/ZnO]n nanolaminate samples. The former two PO’s interaction. models are the result of the different substrates’ interaction.

(a) (b)

FigureFigure 2. GrazingGrazing incidence incidence X X-ray-ray diffraction diﬀraction (GIXRD) (GIXRD) patterns patterns for for ZnO ZnO and and Al2 AlO32 singleO3 single layers, layers, and and[Al2O [Al3/ZnO]2O3/ZnO]n multilayern multilayer (n = 1, (n 2,= 5,1, and 2, 5, 10) and samples 10) samples deposited deposited on (a) on 316L (a) 316LSS and SS ( andb) silicon (b) silicon (100) (100)substrates. substrates.

ReﬁnementRefinement of thethe unit-cell,unit-cell, fullfull widthwidth atat halfhalf maximummaximum (FWHM),(FWHM), andand shapeshape parametersparameters werewere achievedachieved by by the the LeBail-based LeBail-based “pattern “pattern matching” matching” method method implemented implemented in thethe FullprofFullprof program. Likewise,Likewise, the the determination determination of of the the crystallite crystallite size size was was carried carried out by out combining by combining FWHM FWHM peak- peak-analysis—theanalysis—the instrumental instrumental resolution resolution was wasderived derived from from calibration calibration on powdered on powdered Si provided Si provided by byNational National Institute Institute of Standards of Standards and and Technology Technology (NIST (NIST)—using)—using the Debyethe Debye-Scherrer-Scherrer equation, equation, and andsoftware software Jade Jade MDI MDI 9.7 (Table 9.7 (Table 1). 1). The trend to a crystallite-size diminution as n increases was proved from n = 2 to n = 10, for both substrates, Si and 316L SS. This behavior is explained in terms of the diffusivity of Al/Al2O3 into the overlaid crystalline zincite. The extent of atomic diffusion increased with a higher number of interlayer interphases or thinner layers. The subsequent influence on defects-related phenomena (e.g., microstrains or crystalline lattice distortions) with concomitant disruption of crystal growth was evidenced by the broadening of GIXRD peaks [33]. On the other hand, this increase of crystal-growth

Crystals 2020, 10, 620 6 of 16

Table 1. Zincite lattice parameters and crystallite size in both single and multilayers growths on Si(100) wafer and 316L SS.

Lattice Parameters (Å) Substrate Sample Crystallite Size(002) (Å) a = b C Si(100) ZnO 3.2450(4) 5.2044(3) 383(4)

(Al2O3/ZnO)1 3.2416(8) 5.2028(7) 197(4)

(Al2O3/ZnO)2 3.2397(8) 5.2018(1) 267(6)

(Al2O3/ZnO)5 3.2467(13) 5.2042(7) 179(4)

(Al2O3/ZnO)10 3.2569(19) 5.1988(11) 93(3) 316L SS ZnO 3.2530(23) 5.2163(24) 428(9)

(Al2O3/ZnO)1 3.265(122) 5.2166(31) 226(4)

(Al2O3/ZnO)2 3.2492(35) 5.2156(12) 221(8)

(Al2O3/ZnO)5 3.2558(55) 5.2208(15) 175(6)

(Al2O3/ZnO)10 3.2662(165) 5.2186(32) 79(3) () Estimated Standard Deviation—ESD.

The trend to a crystallite-size diminution as n increases was proved from n = 2 to n = 10, for both substrates, Si and 316L SS. This behavior is explained in terms of the diffusivity of Al/Al2O3 into the overlaid crystalline zincite. The extent of atomic diffusion increased with a higher number of interlayer interphases or thinner layers. The subsequent influence on defects-related phenomena (e.g., microstrains or crystalline lattice distortions) with concomitant disruption of crystal growth was evidenced by the broadening of GIXRD peaks [33]. On the other hand, this increase of crystal-growth disruptions phenomena was not observed when n = 2 nanolaminate samples are compared with the n = 1 counterpart. The crystallite size in n = 2 showed a higher value in the case of samples with a silicon substrate, 267 Å (i.e., a percentage increase of 36%). It practically remained unchanged for the steel-substrate sample, 221 Å. The first-deposited Al2O3 layer appears here as a diffusion barrier when n = 2; its relatively high thickness (i.e., ~ 52 nm) compromised diffusivity effects and, consequently, precluded the occurrence of crystalline defects. The experimental design to growth nanolaminates plays a crucial role in this discussion since the total thickness of the different-n nanolaminates remains constant (i.e., ~210 nm). The proper thickness of Al2O3 or ZnO layers varied in each growing experiment. Taking into consideration that the experiments were conducted on selected values of n (1, 2, 5, and 10), the 52 nm-thickness of the first-deposited Al2O3 layer in the [Al2O3/ZnO]2 nanolaminate could mark a behavior threshold from which (in terms of n, for n 2), the crystallite ≥ size of wurtzite-type ZnO decreases. The crystallite sizes when n = 1—i.e., 197 and 226 Å for growths on silicon single-crystal wafer and 316L stainless steel, respectively—were notably inferior to those without overlying Al2O3. Although this behavior indicates ZnO growth rates higher in nanolaminates than in single layers growth on the primary substrates, former cases were mainly governed by the diffusion effects mentioned above. Regardless the particular crystallite-size of zincite in [Al2O3/ZnO]n nanolaminates, it is worth underlining that the outstanding highest values (see Table1) correspond to the ZnO layer deposited directly on the primary substrates, 383 and 428 Å for Si and 316L SS, respectively. These values are comparable with those from ZnO film growth by different techniques reported elsewhere: spray pyrolysis, ultrasonic spray, and pulsed laser deposition [34–36].

3.2. X-ray Photoelectron Spectroscopy Analysis The chemical composition and binding energy of the elements were determined with X-ray photoelectron spectroscopy (XPS). Figure3a,b show the XPS survey scans from Al 2O3 and ZnO samples, Crystals 2020, 10, x 7 of 16

correspond to the high-resolution spectra associate with the ZnO layer. The first signal was assigned to the O 1s and the second signal corresponds to Zn 2p3/2 element. The ZnO was adjusted with two peaks, the first one is located around 1020.7 eV and the second one is located around 1022.6 eV Crystalsattributed2020, 10 to, 620 Zn 2p3/2 [32,40]. Considering the last discussion, it was possible to determine that7 of the 16 stoichiometric relations for alumina- and zinc oxide-obtained layers correspond approximately to Al0.4O0.6 and Zn0.4O0.6, respectively. The fact that the surface of ZnO contains oxygen in a concentration respectively, displaying the signals of the elements on their surfaces. The more intense signals are due higher than one-to-one with Zn could be due to environmental contamination during transporting to the O, Al, and Zn elements; even so, traces of carbon are present. from preparation to XPS measurement.

(a) (b)

(e) (f)

FigureFigure 3. 3.XPS XPS survey survey spectra spectra and and high-resolution high-resolution signals signals for for single single layers: layers: (a ()a survey) survey spectra spectra AlxO1 AlxO1x,−x, − (b) survey spectra Zn-O, (c) Al 2p, (d) O 1s (Al2O), (e) Zn (2p3) signals and (f) O 1s signal (ZnO). (b) survey spectra Zn-O, (c) Al 2p, (d) O 1s (Al2O), (e) Zn (2p3) signals and (f) O 1s signal (ZnO).

3.3. FigureAtomic3 cForce shows Microscopy the high-resolution (AFM) XPS spectra of the Al 2O3 single layer, evidencing the oxygen signal (O 1s). Similarly, Figure3d shows the high-resolution spectra corresponding to aluminum Figure 4 exhibits the AFM surface topography associated with single layers and multilayers in the Al 2p region. The Al 2p signal related to Al O has been observed with a binding energy of deposited on the 316L SS substrate, which shows 2the3 cluster formations. It can be observed that the 74.5 eV [37]. This particular binding energy position corresponds to the Al O phase formation [38]. morphological shapes on the samples surface and height surface decreases2 3 with the increase of The high-resolution signal of the O 1s was found at a binding energy of 533 eV [39]. Figure3c–f shows the experimental data as scatter plots and solid lines which are the best ﬁt for two Gaussian functions; the deconvoluted plots are also shown in these graphs. Figure3e,f correspond to the high-resolution spectra associate with the ZnO layer. The ﬁrst signal was assigned to the O 1s and the second signal corresponds to Zn 2p3/2 element. The ZnO was adjusted with two peaks, Crystals 2020, 10, 620 8 of 16

the ﬁrst one is located around 1020.7 eV and the second one is located around 1022.6 eV attributed to Zn 2p3/2 [32,40]. Considering the last discussion, it was possible to determine that the stoichiometric relations for alumina- and zinc oxide-obtained layers correspond approximately to Al0.4O0.6 and Zn0.4O0.6, respectively. The fact that the surface of ZnO contains oxygen in a concentration higher than one-to-one with Zn could be due to environmental contamination during transporting from preparation to XPS measurement.

3.3. Atomic Force Microscopy (AFM) Figure4 exhibits the AFM surface topography associated with single layers and multilayers deposited on the 316L SS substrate, which shows the cluster formations. It can be observed that the Crystalsmorphological 2020, 10, x shapes on the samples surface and height surface decreases with the increase of bilayer8 of 16 numbers. This means a gradual decrease in roughness values. Moreover, the clusters and roughness in roughnessFigure4c forin theFigure [Al 4c2O 3for/ZnO] the 2[Alnanolaminates2O3/ZnO]2 nanolaminates present a combination present a ofcombination the growth of of the Al2 growthO3 on ZnO of Allayers2O3 on [10 ZnO,41]. layers [10,41].

Figure 4. Surface topography for (a) ZnO, (b) Al2O3 single layers and (c) [Al2O3/ZnO]1, Figure 4. Surface topography for (a) ZnO, (b) Al2O3 single layers and (c) [Al2O3/ZnO]1, (d) (d) [Al2O3/ZnO]2, and (e) [Al2O3/ZnO]5 multilayers. [Al2O3/ZnO]2, and (e) [Al2O3/ZnO]5 multilayers. Figure5 shows the reduction of the average roughness with the increase in the bilayer numbers. AccordingFigure 5 to shows the restriction the reduction model, of the this average is a behavior roughness presented with the by increase the multilayers in the bilayer of a superlattice, numbers. Abecauseccording the to increasethe restriction in the number of bilayers with a constant total thickness represents a decrease in the bilayermodel, period,this is a and behavior an increase presented in the numberby the multilayers of the interfaces of a superlattice, due to the decrease because of the the increase thickness in of thethe number bilayer. of Figure bilayers5 also with shows a constant that the total grain thickness size decreases represents with thea decrease increase in of the the bilayer number period, of bilayers. and anThe increase grain sizein the for number the ZnO of single the interfaces layer was due 89.3 to nm, the but decrease we could of the not thicknes obtain thiss of value the bilayer. for multilayers Figure 5from also shows the AFM that measurements the grain size becausedecreases the with AFM the measurements increase of the were number carried of bilayers. out on the The surface grain ofsize the formultilayers, the ZnO single where layer the upper was 89.3 layer nm, was but always we could the amorphous not obtain layerthis value of Al2 Ofor3. multilayers from the AFM measurements because the AFM measurements were carried out on the surface of the multilayers, where the upper layer was always the amorphous layer of Al2O3.

3.0

2.5 2.4 Roughness 2.0 1.8 1.5 1.2 1.0 ( nm 0.6 ) Grain Size (nm)SizeGrain 0.5

0.0 0.0 ZnO Al2O3 n= 1 n= 2 n= 5 n= 10 Samples

Figure 5. Roughness and grain size evolution of ZnO and Al2O3 single layers, and [Al2O3/ZnO]n bilayers with the increase of bilayers number.

Crystals 2020, 10, x 8 of 16 roughness in Figure 4c for the [Al2O3/ZnO]2 nanolaminates present a combination of the growth of Al2O3 on ZnO layers [10,41].

Figure 4. Surface topography for (a) ZnO, (b) Al2O3 single layers and (c) [Al2O3/ZnO]1, (d) [Al2O3/ZnO]2, and (e) [Al2O3/ZnO]5 multilayers.

Figure 5 shows the reduction of the average roughness with the increase in the bilayer numbers. According to the restriction model, this is a behavior presented by the multilayers of a superlattice, because the increase in the number of bilayers with a constant total thickness represents a decrease in the bilayer period, and an increase in the number of the interfaces due to the decrease of the thickness of the bilayer. Figure 5 also shows that the grain size decreases with the increase of the number of bilayers. The grain size for the ZnO single layer was 89.3 nm, but we could not obtain this value for multilayers from the AFM measurements because the AFM measurements were carried out on the surface of the multilayers,Crystals 2020, 10 where, 620 the upper layer was always the amorphous layer of Al2O3. 9 of 16

3.0

2.5 2.4 Roughness 2.0 1.8 1.5 1.2 1.0 ( nm 0.6 ) Grain Size (nm)SizeGrain 0.5

0.0 0.0 ZnO Al2O3 n= 1 n= 2 n= 5 n= 10 Samples

Figure 5. Roughness and grain size evolution of ZnO and Al2O3 single layers, and [Al2O3/ZnO]n CrystalsFigurebilayers 2020, 10 5., with xRoughness the increase and of grain bilayers size number. evolution of ZnO and Al2O3 single layers, and [Al2O3/ZnO]9n of 16 bilayers with the increase of bilayers number. 3.4.3.4. TEM TEM A Analysisnalysis TransmissionTransmission electron electron microscopy microscopy (TEM) (TEM) micrographs micrographs in the in cross-sectional the cross-sectional mode were mode obtained were obtainedto validate to and validate verify and the total verify multilayer the total thickness. multilayer Figure thickness.6a presents Figure the 6a TEM presents cross-sectional the TEM view cross of- sectional[Al2O3/ZnO] view10 ofmultilayers, [Al2O3/ZnO] and10 Figuremultilayers,6b shows and a Fig higherure 6b resolution shows a image higher of resolution two Al 2O 3image/ZnO bilayers,of two Alwhere2O3/ZnO it was bilayers, possible where to corroborate it was possible the multilayersto corroborate arrangement the multilayers formation arrangement with 11.1 formation and 12.0 with nm 11.1thickness and 12.0 for nm each thickness ZnO and for Aleach2O 3ZnOmonolayers, and Al2O3 respectively, monolayers, andrespectively, an average and total an average thickness total of thicknessapproximately of approximately 210.0 nm. A210.0 change nm. inA change the shade in the of theshade gray of colorthe gray could color be c observed,ould be observed, which is whichassociated is associated with a change with ina change the electronic in the densityelectronic of the density materials of the in thematerials multilayer in the stack. multilayer Therefore, stack. the Therefore,modulation the of modulationthe grayscale of change the grayscale is associated change with is di associatedﬀerent layers, with where different the darker layers, gray where color the is darkerrelated gray to the color ZnO is layer. related From to image the ZnO 6b, ZnOlayer. layer From shows image (002) 6b, interplanar ZnO layer distances shows (002) around interplanar 0.260 nm, distanceswhich is very around close 0.260 to that nm, in whichthe JCPDS is very card close 36-1451, to that as well in the as in JCPDS [32,41 , card42]. The 36-1451, amorphous as well growth as in [32,41,42]of the Al2. OThe3 layer amorphous is evident growth in the of same the Al ﬁgure.}2O3 layer is evident in the same figure.}

(a) (b)

FigureFigure 6. 6. TEMTEM cross cross-sectional-sectional views views of of [Al [Al22OO3/ZnO]3/ZnO]n nbilayersbilayers (a ()a ) nn == 1010 and and (b (b) )higher higher resolution resolution imageimage of of an an Al Al22OO3/3ZnO/ZnO bilayer. bilayer. 3.5. Electrochemical Analysis 3.5. Electrochemical Analysis

The electrochemical behavior of the [Al2O3/ZnO]n/316L system was determined by the The electrochemical behavior of the [Al2O3/ZnO]n/316L system was determined by the electrochemical impedance spectroscopy technique in the presence of Hank´s balanced salt electrochemical impedance spectroscopy technique in the presence of Hank´s balanced salt solution solution (HBSS). Figure7a displays the Nyquist diagram, the imaginary part of the impedance versus its (HBSS). Figure 7a displays the Nyquist diagram, the imaginary part of the impedance versus its real real part, for the 316L stainless steel, ZnO, and Al2O3 single layers and [Al2O3/ZnO]n/316L multilayers. part, for the 316L stainless steel, ZnO, and Al2O3 single layers and [Al2O3/ZnO]n/316L multilayers. Figure7b shows the equivalent circuit [ 43] simulating the substrate-coating and coating-electrolyte Figure 7b shows the equivalent circuit [43] simulating the substrate-coating and coating-electrolyte interfaces, as a double layer capacitance in parallel with the coating resistance and the electrolyte resistance due to the ion transfer reaction from the electrolyte to the metallic substrate [44].

(a) (b)

Figure 7. (a) Impedance diagrams of 316L stainless steel, ZnO and Al2O3 single layers and [Al2O3/ZnO]n/316L multilayers. The inset shows the Nyquist diagram of 316L SS. (b) Equivalent

Crystals 2020, 10, x 9 of 16

3.4. TEM Analysis Transmission electron microscopy (TEM) micrographs in the cross-sectional mode were obtained to validate and verify the total multilayer thickness. Figure 6a presents the TEM cross- sectional view of [Al2O3/ZnO]10 multilayers, and Figure 6b shows a higher resolution image of two Al2O3/ZnO bilayers, where it was possible to corroborate the multilayers arrangement formation with 11.1 and 12.0 nm thickness for each ZnO and Al2O3 monolayers, respectively, and an average total thickness of approximately 210.0 nm. A change in the shade of the gray color could be observed, which is associated with a change in the electronic density of the materials in the multilayer stack. Therefore, the modulation of the grayscale change is associated with different layers, where the darker gray color is related to the ZnO layer. From image 6b, ZnO layer shows (002) interplanar distances around 0.260 nm, which is very close to that in the JCPDS card 36-1451, as well as in [32,41,42]. The amorphous growth of the Al2O3 layer is evident in the same figure.}

(a) (b) Crystals 2020, 10, x 10 of 16 Figure 6. TEM cross-sectional views of [Al2O3/ZnO]n bilayers (a) n = 10 and (b) higher resolution image of an Al2O3/ZnO bilayer. circuits used for simulation of the experimental data, where RE, RS, RP, CPE and WE are the reference 3.5. Electrochemicalelectrode, electrolyte Analysis resistance, polarization resistance of coatings, the constant elements of phase, and the working electrode (the sample), respectively. The electrochemical behavior of the [Al2O3/ZnO]n/316L system was determined by the electrochemicalFrom the impedance Nyquist diagrams spectroscopy and the technique equivalent in circuithe presencet (Figure of 7b), Hank´s the values balanced for the salt polarization solution (HBSS).Crystalsresistance Fig2020ure, 10 (R ,7a 620p) displays were extracted. the Nyquist The diagram, results presentthe imaginary in Fig urepart 8bof the indicate impedance that theversus polarization its 10real of 16 part,resistance for the increase316L stainlessd with steel,the increase ZnO, and of the Al bilayer2O3 single number, layers confirming and [Al2O the3/ZnO] protectiven/316L multilayers.effect of layers Figandure mult7b showsilayers. the equivalent circuit [43] simulating the substrate-coating and coating-electrolyte interfaces,interfaces,Fig ureas as a8a double a shows double layer the layer Tafel capacitance capacitance polarization in inparallel parallelcurves, with withwhere the the thecoating coating corrosion resistance resistance potential and and theis thedisplayed electrolyt electrolyte ase a resistance due to the ion transfer reaction from the electrolyte to the metallic substrate [44]. resistancefunction due of the to thelogarithm ion transfer of the reaction corrosion from current the electrolyte density for to the the 316L metallic stainless substrate steel, [ ZnO44]. , and Al2O3 single layers and [Al2O3/ZnO]n/316L multilayers. The curves for ZnO and Al2O3 single layers and [Al2O3/ZnO]n/316L multilayers were in the negative region of the corrosion potential. The above indicates that in any case there was ion transfer from the electrolyte to the metallic substrate, but it was less negative than for the 316L substrate, and it decreased with the increase in the [Al2O3/ZnO]n bilayer number. The slopes of the anodic and cathodic zones and the plateau region of the Tafel curves gave information about the corrosion rate. The plateau value increased strongly for the single layers and multilayers in comparison to that value from the uncoated 316L substrate, indicating that the coatings provided greater corrosion protection compared to uncoated 316L SS surface. Tafel polarization curves were used to calculate the corrosion current density icorr using the Stern-Geary model, with the Equation (4) [45].

= (4) (a) 2,03( + ) (b)

2 3 WhereFigureFigure β a 7.and 7.(a ) β(ac Impedance )are Impedance the Tafel, diagrams diagrams anodic of and of 316L 316L cathodic stainless stainless slopes steel, steel, ZnOrespectively, ZnO and and Al Al Oand2Osingle3 singleRp is layers the layers polarization and and resistance[Al[Al2O23/ZnO]O3 value/ZnO]n/316L nobtained/316L multilayers. multilayers. from the The TheNyquist inset inset shows diagram. shows the the Nyquist However, Nyquist diagram diagram the corrosion of of316L 316L SS. current SS. (b) ( b Equivalent) Equivalentcan be directly relatedcircuits to the used corrosion for simulation rate through of the experimental the following data, E wherequation RE, (5) RS,: RP, CPE and WE are the reference

electrode, electrolyte resistance, polarization resistance of coatings, the constant elements of phase, and the working electrode (the sample), respectively.= 0,13 ( ) (5)

Where:From ρ is the the Nyquist density diagrams of the material and the in equivalentg/cm3, We is circuit the electrochemical (Figure7b), the equivalent values for theof the polarization molecular resistanceweight of the (Rp metal) were divided extracted. by the The number results of electrons present in involved Figure8 inb the indicate anodic that reaction. the polarization Finally, 0.13 resistanceis a constant increased that includes with the the increase Faraday of the constant bilayer andnumber, the cconﬁrmingonversion the factor protective necessary eﬀect to of give layers the andcorrosion multilayers. rate in micrometer per year (µm/y) when icorr is expressed in µA/m2.

(a) (b)

FigureFigure 8.8. ((aa)) Tafel Tafel polarization polarization curves. curves. ( (bb)) Polarization Polarization resistance resistance ( (RRpp) andand corrosion corrosion rate, rate, of of 316L 316L stainlessstainless steel,steel, ZnO,ZnO, andand AlAl22OO33 singlesingle layers and [Al 2OO33/ZnO]/ZnO]nn/316L/316L multilayers. multilayers.

FigureThe results8a shows presented the Tafel in polarizationFigure 8b indicate curves, that where the thepolarization corrosion resistance potential increase is displayedd and as the a functioncorrosion of rate the diminishe logarithmd of when the corrosion the 316L currentSS substrate density wa fors covered the 316L with stainless ZnO and steel, Al ZnO,2O3 single and Allayers2O3 singleand with layers the and increase [Al2O of3/ZnO] the [Aln/316L2O3/ZnO] multilayers.n bilayer Thenumber, curves confirming for ZnO and the Al protective2O3 single effect layers of and the [Allayers2O3 and/ZnO] multilayersn/316L multilayers against corrosion. were in theThe negativereason for region this protective of the corrosion effect is potential.related to the The fa abovect that indicates that in any case there was ion transfer from the electrolyte to the metallic substrate, but it was less negative than for the 316L substrate, and it decreased with the increase in the [Al2O3/ZnO]n bilayer number. The slopes of the anodic and cathodic zones and the plateau region of the Tafel curves gave information about the corrosion rate. The plateau value increased strongly for the single layers and multilayers in comparison to that value from the uncoated 316L substrate, indicating that the coatings provided greater corrosion protection compared to uncoated 316L SS surface. Tafel polarization Crystals 2020, 10, 620 11 of 16

curves were used to calculate the corrosion current density icorr using the Stern-Geary model, with the Equation (4) [45]. βaβc icorr = (4) 2.03Rp(βa + βc) where βa and βc are the Tafel, anodic and cathodic slopes respectively, and Rp is the polarization resistance value obtained from the Nyquist diagram. However, the corrosion current can be directly related to the corrosion rate through the following Equation (5): ! We V = 0.13 (icorr) (5) corrosion ρ

3 where: ρ is the density of the material in g/cm , We is the electrochemical equivalent of the molecular weight of the metal divided by the number of electrons involved in the anodic reaction. Finally, 0.13 is a constant that includes the Faraday constant and the conversion factor necessary to give the corrosion 2 rate in micrometer per year (µm/y) when icorr is expressed in µA/m . The results presented in Figure8b indicate that the polarization resistance increased and the corrosion rate diminished when the 316L SS substrate was covered with ZnO and Al2O3 single layers and with the increase of the [Al2O3/ZnO]n bilayer number, confirming the protective effect of the layers and multilayers against corrosion. The reason for this protective effect is related to the fact that the ZnO crystallites become smaller as the number of multilayers increases, and therefore the total number of grain boundaries in the ZnO material grows larger, giving a higher polarization resistance.

3.6. Lactate Dehydrogenase Test Analysis The calculated values for cytotoxicity determined according to Equation (3) are shown in Figure9b for the ZnO and Al2O3 single layers, [Al2O3/ZnO]n multilayer system, and the 316L SS. According to ISO 10993-531 [46,47] the results may show a low level of cytotoxicity or grade “2”, which means a slight reactivity for the qualitative morphological qualification of the cytotoxicity of the sample, when the percentage of cytotoxicity does not exceed 60%; and a degree “1” or light reactivity when it has a value lower than 20%. The grade “0” will be for materials or extracts without reactivity. Based on this, we observed at 24 h that 316 SS was almost at the limit of mild reactivity with 51.3%; therefore, for short time applications, it could give a good response, but for longer terms, lower cytotoxicity is desirable. Al2O3 single layer exhibited similar behavior as 316L SS, with 40.3% of cytotoxicity given that the Al2O3 single layer is amorphous. A very different behavior was observed for ZnO single layer, which is crystalline and has the largest crystallite size (428 9 Å). In this case, the cytotoxicity percentage ± was 1.6%, which is almost not reactive; this could mean either that ZnO has the lowest lysis effect in CHO cells for 24 h or that it does not interfere with the cell culture growth. In the case of the multilayers, with n = 1 and n = 2, they presented a lower cytotoxicity behavior with a slight reactivity (grade “1”), a cytotoxicity percentage of 29.8% and 30.3%, respectively. This means that occasional lysed cells are present during assay, so few cell populations were damaged. While for n = 5 and n = 10, a mild reactivity was assumed, and they showed a cytotoxicity percentage of 38.5% and 38.0%, respectively. The cytotoxicity dependence behavior of the samples with the number of bilayers is related to several factors, such as the ZnO/Al2O3 bilayers thickness, the crystallite size of the ZnO layer, and the roughness and amorphicity of the Al2O3 upper layer. Figure9b shows the correlation between these factors. We can conclude that for [Al2O3/ZnO]n/316L multilayer structure, the samples with n = 1 and n = 2, ZnO’s higher layer thicknesses (100 and 50 nm) and crystallite sizes (226 and 221 Å, respectively) show the lowest cytotoxicity compared to multilayers with a greater bilayer number (n = 5 and 10), where the ZnO thicknesses and crystallite size are smaller (175 and 79 Å, respectively). The above confirms the important role that the ZnO layer plays. Crystals 2020, 10, x 11 of 16

the ZnO crystallites become smaller as the number of multilayers increases, and therefore the total number of grain boundaries in the ZnO material grows larger, giving a higher polarization resistance.

3.6. Lactate Dehydrogenase Test Analysis The calculated values for cytotoxicity determined according to Equation (3) are shown in Figure 9b for the ZnO and Al2O3 single layers, [Al2O3/ZnO]n multilayer system, and the 316L SS. According to ISO 10993-531 [46,47] the results may show a low level of cytotoxicity or grade “2”, which means a slight reactivity for the qualitative morphological qualification of the cytotoxicity of the sample, when the percentage of cytotoxicity does not exceed 60%; and a degree “1” or light reactivity when it has a value lower than 20%. The grade “0” will be for materials or extracts without reactivity. Based on this, we observed at 24 h that 316 SS was almost at the limit of mild reactivity with 51.3%; therefore, for short time applications, it could give a good response, but for longer terms, lower cytotoxicity is desirable. Al2O3 single layer exhibited similar behavior as 316L SS, with 40.3% of cytotoxicity given that the Al2O3 single layer is amorphous. A very different behavior was observed for ZnO single layer, which is crystalline and has the largest crystallite size (428 ± 9 Å). In this case, the cytotoxicity percentage was 1.6%, which is almost not reactive; this could mean either that ZnO has the lowest lysis effect in CHO cells for 24 hr or that it does not interfere with the cell culture growth. In the case of the multilayers, with n = 1 and n = 2, they presented a lower cytotoxicity behavior with a slight reactivity (grade “1”), a cytotoxicity percentage of 29.8% and 30.3%, respectively. This means that occasional lysed cells are present during assay, so few cell populations were damaged. CrystalsWhile 2020for ,n10 =, 5 620 and n = 10, a mild reactivity was assumed, and they showed a cytotoxicity percentage12 of 16 of 38.5% and 38.0%, respectively.

(a) (b)

FigureFigure 9. 9. ((aa)) Citotoxicity percentage for 316L 316L stainless stainless steel, steel, ZnO, ZnO, and and Al Al22OO33single layers layers and and [Al[Al22O3/ZnO]/ZnO]n/316L/316L multilayers. multilayers. ( b) (b [Al) 2[AlO32/OZnO]3/ZnO]n/316Ln/316L cytotoxicity cytotoxicity behavior behavior as a function as a of function roughness of ofroughness top Al2O of3 layer, top Al and2O3 crystallitelayer, and sizecrystallite and thickness size and of thickness the ZnO of layer. the ZnO layer.

Summarizing,The cytotoxicity the dependence LDH test for behavior all samples of the showed samples low with cytotoxicity the number levels of under bilayers 60%, is approvedrelated to forseveral short factors terms, applications.such as the ZnO/Al2O3 bilayers thickness, the crystallite size of the ZnO layer, and the Crystals 2020, 10, x 12 of 16 roughnessFigure and 10 presents amorphicity the cell of adhesionthe Al2O3 SEMupper images layer. forFigure 316L 9b/ZnO shows and the 316L correlation/Al2O3 monolayers between these and behavior316Lfactors./[Al We2 Oto3 canthe/ZnO] surfaceconcluden multilayers was that closely for (n [Al= linked1,2O 2,3/ZnO] 5, andto then/316L 10). level In multilayer these of cytotoxicity images structure, it can of bethe the seen samples samples that the (see with cell Figure adhesionn = 1 9anda). behavior to the surface was closely linked to the level of cytotoxicity of the samples (see Figure9a). Onn = the 2, other ZnO’s hand, higher except layer for thicknesses the 316L/[Al (1002O 3and /ZnO] 5010 nm)multilayers, and crystallite it can be sizes observed (226 andthat 221on the Å, surfaceOnrespectively) the otherof the show hand,multilayers, the except lowest groups for cytotoxicity the of 316L cells/[Al were compared2O3 close/ZnO]ly to10 linked multilayersmultilayers, between with it and can a greater to be the observed surface, bilayer that numberextending on the (n surface of the multilayers, groups of cells were closely linked between and to the surface, extending the= 5 fineand anchor10), where joints the towards ZnO thicknesse it, which sallowed and crystallite their correct size are fixing. smaller This (175 may and be due 79 Å, to respectively)the Al2O3 top. layertheThe ﬁne above surface anchor confirms roughness joints the towards (lessimportant than it, which 1 role nm) allowedthat for allthe samples ZnO their layer correct in the plays. ﬁxing. multilayer, This maywhich be meant due to less the anchori Al2O3 topng flayeror cellsSummarizing, surface that needed roughness the it. LDH (less test than for 1 nm) all samples for all samples showed in low the cytotoxicity multilayer, whichlevels under meant 60%, less anchoringapproved forfor cellsshort that terms needed applications. it. Figure 10 presents the cell adhesion SEM images for 316L/ZnO and 316L/Al2O3 monolayers and 316L/[Al2O3 /ZnO]n multilayers (n = 1, 2, 5, and 10). In these images it can be seen that the cell adhesion ZnO Al2O3 n = 1

n = 2 n = 5 n = 10

Figure 10. Cell adhesion scattering electron microscopy (SEM) images for ZnO and Al2O3 single layers Figure 10. Cell adhesion scattering electron microscopy (SEM) images for ZnO and Al2O3 single layers and 316L/[Al2O3 /ZnO]n multilayers (n = 1, 2, 5, and 10). and 316L/[Al2O3 /ZnO]n multilayers (n = 1, 2, 5, and 10).

Thus, if the CHO cells cannot adhere to a surface, they tend to disperse, floating on the fluid. Within a given time, cells need other cells to interact with them and survive as a group: if this does not happen, cell junctions break and they die. This does not mean samples were toxic, but that these surfaces are not adherent and could be used in applications such as acetabular prosthesis or for femoral head, where friction will be necessary to obtain normal activity for the patient. For the sample of ZnO we did obtain a different behavior, since the CHO cells got anchored to the surface, which lead to a good cell interaction and union between them. This behavior develops a net of well-knitted cells, which will grow to confluence. The ZnO layer probably leads to a better adherent surface because its roughness is higher than Al2O3 roughness and gives more anchored points for the fixation of the cells. These kinds of surfaces could be used in other applications, for example, for femoral prosthesis replacement.

4. Conclusions

Crystals 2020, 10, 620 13 of 16

Thus, if the CHO cells cannot adhere to a surface, they tend to disperse, floating on the fluid. Within a given time, cells need other cells to interact with them and survive as a group: if this does not happen, cell junctions break and they die. This does not mean samples were toxic, but that these surfaces are not adherent and could be used in applications such as acetabular prosthesis or for femoral head, where friction will be necessary to obtain normal activity for the patient. For the sample of ZnO we did obtain a different behavior, since the CHO cells got anchored to the surface, which lead to a good cell interaction and union between them. This behavior develops a net of well-knitted cells, which will grow to confluence. The ZnO layer probably leads to a better adherent surface because its roughness is higher than Al2O3 roughness and gives more anchored points for the fixation of the cells. These kinds of surfaces could be used in other applications, for example, for femoral prosthesis replacement.

4. Conclusions

The structure, chemical bonds, morphology, and electrochemical properties of ZnO and Al2O3 single layers and the [Al2O3/ZnO]n nanolaminates system deposited on 316L SS, along with additional growths on Si(100) wafer for comparison, were studied to elucidate critical factors that determine their cytotoxicity and subsequent biocompatibility. An amorphous alumina layer was always deposited overlaying on the zincite crystalline-type ZnO-layer of the [Al2O3/ZnO]n stack motifs of the nanolaminates. ZnO formed in [Al2O3/ZnO]n multilayers showed a decrease in its crystallite size due to the influence of the alumina that induces crystalline defects evidenced by a broadening of ZnO GIXRD-peaks (i.e., changes in FWHM), as well as a persistent preferred orientation on (002). Otherwise, the ZnO single layers exhibited the largest crystallite size. Concerning this behavior of crystallite size, no significant differences were appreciated between samples deposited on 316L SS or Si(100) substrates. AFM morphological analysis demonstrated that the grain size and roughness on the surface decreased as the [Al2O3/ZnO]n bilayer number increased (for n > 2), directly correlated with the concomitant decrease of crystallite size in ZnO. Finally, the electrochemical study showed that corrosion resistance of [Al2O3/ZnO]n nanolaminates was higher in comparison with the 316L SS surface, and ZnO, and Al2O3 single layer, confirming the protective effect of the layers and multilayers against corrosion. This protective effect is because the ZnO crystallites become smaller as the number of multilayers increases, and therefore the total number of grain boundaries in the ZnO material grows larger, resulting in a higher polarization resistance. According to LDH in CHO cells, the biocompatible behavior showed low cytotoxic responses. This means that the ZnO single layer had an optimal response because its LDH release was minimum, associated with the best crystallinity of this single layer, which generates a higher roughness than multilayers. Additionally, the highest cytotoxicity, after Cu (reference) and 316L SS, was for the Al2O3 single layer, due to its amorphous nature and a relatively high corrosion rate. So, considering that the top layer is Al2O3, the multilayers would not present a viable solution for biocompatibility applications because of its high cytotoxicity. This work demonstrated that nanolaminates reduced from high to medium cytotoxicity grade. To improve the cytotoxic response and biocompatibility of [Al2O3/ZnO]n/316L nanolaminates, we recommend changing the top-layer Al2O3 for ZnO ([ZnO/Al2O3]n multilayer system) in samples with n = 1 and n = 2. This method is a very reliable one to produce continuous and fine layers with uniform properties on the surface they are applied to. Nevertheless, its cost is high and limited in the thickness that can be obtained, although this is compensated for by using a more economical substrate in biomedical applications.

Author Contributions: Conceptualization, H.T., J.C.C. and G.Z.; Data curation, D.O., J.L., M.A.H.-L., M.R.-C. and G.Z.; Formal analysis, D.O., M.A.H.-L. and M.R.-C.; Funding acquisition, H.T., M.H.F., J.M.Y.-L. and G.Z.; Investigation, D.O., J.L., M.H.F., J.M.Y.-L., J.O.G., J.C.C. and G.Z.; Methodology, G.Z.; Project administration, G.Z.; Resources, J.L. and J.O.G.; Supervision, G.Z.; Validation, J.L., M.A.H.-L., M.R.-C., J.O.G. and G.Z.; Visualization, G.Z.; Writing—original draft, D.O., J.L., H.T., M.H.F., M.A.H.-L., M.R.-C., J.M.Y.-L. Crystals 2020, 10, 620 14 of 16 and G.Z.; Writing—review & editing, G.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by funds from internal calls for projects at “Universidad del Valle”, Cali, Colombia 2015 (CI 71023), Basic Science projects 2017-2018 A1-S-21084, FORDECYT-CONACYT 272894 project and DGAPA-UNAM, through research projects: PAPIIT IN103220, IN112117, IN110018, IN113219, and the Center of Excellence for Novel Materials (CENM), Universidad del Valle. Special acknowledgments to COLCIENCIAS support through the program of “Jovenes Investigadores e Innovadores 2016” (FP44842-078-2017). The authors are grateful for the facilities granted in the use of the LIDTRA experimental infrastructure through the projects of national laboratories of CONACYT, LN295261, and LN254119. Acknowledgments: This work was partially supported by funds from internal calls for projects at “Universidad del Valle”, Cali, Colombia 2015 (CI 71023), Basic Science projects 2017-2018 A1-S-21084, FORDECYT-CONACYT 272894 project and DGAPA-UNAM, through research projects: PAPIIT IN103220, IN112117, IN110018, IN113219, and the Center of Excellence for Novel Materials (CENM), Universidad del Valle. Special acknowledgments to COLCIENCIAS support through the program of “Jovenes Investigadores e Innovadores 2016” (FP44842-078-2017) The authors are grateful for the facilities granted in the use of the LIDTRA experimental infrastructure through the projects of national laboratories of CONACYT, LN295261, and LN254119. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Tlili, B.; Nouveau, C.; Walock, M.; Nasri, M.; Ghrib, T. Effect of layer thickness on thermal properties of multilayer thin films produced by PVD. Vacuum 2012, 86, 1048–1056. [CrossRef] 2. Ott, A.W.; Klaus, J.W.; Johnson, J.M.; George, S.M.; Oro, L.A. Al3O3 thin film growth on Si (100) using binary reaction sequence chemistry. Thin Solid Films 1997, 292, 135–144. [CrossRef]

3. Elam, J.W.; George, S.M. Growth of ZnO/Al2O3 alloy ﬁlms using atomic layer deposition techniques. Chem. Mater. 2003, 15, 1020–1028. [CrossRef]

4. Elam, J.W.; Sechrist, Z.A.; George, S.M. ZnO/Al2O3 nanolaminates fabricated by atomic layer deposition: Growth and surface roughness measurements. Thin Solid Films 2002, 414, 43–55. [CrossRef] 5. Tirado, S. Thin Films of Zinc Oxide Doped with Aluminium and Fluoride Prepared by Sol-Gel. Av. Cienc. Igenieria 2012, 3, 87–96. 6. López, J.; Martínez, J.; Abundiz, N.; Domínguez, D.; Murillo, E.; Castillón, F.F.; Machorro, R.; Farías, M.H.;

Tiznado, H. Thickness eﬀect on the optical and morphological properties in Al2O3/ZnO nanolaminate thin ﬁlms prepared by atomic layer deposition. Superlattices Microstruct. 2016, 90, 265–273. [CrossRef] 7. Lopez, J.; Solorio, E.; Borbón-Nuñez, H.A.; Castillón, F.F.; Machorro, R.; Nedev, N.; Farías, M.H.; Tiznado, H.

Refractive index and bandgap variation in Al2O3-ZnO ultrathin multilayers prepared by atomic layer deposition. J. Alloys Compd. 2017, 691, 308–315. [CrossRef] 8. Liu, H.; Yang, R.B.; Guo, S.; Lee, C.J.J.; Yakovlev, N.L. Eﬀect of annealing on structural and optical properties

of ZnO/Al2O3 superlattice structures grown by atomic layer deposition at 150 ◦C. J. Alloys Compd. 2017, 703, 225–231. [CrossRef] 9. Homola, T.; BuršÝkovß, V.; Ivanova, T.V.; Souˇcek,P.; Maydannik, P.S.; Cameron, D.C.; Lackner, J.M.

Mechanical properties of atomic layer deposited Al2O3/ZnO nanolaminates. Surf. Coat. Technol. 2015, 284, 198–205. [CrossRef] 10. Chaaya, A.A.; Viter, R.; Baleviciute, I.; Bechelany, M.; Ramanavicius, A.; Gertnere, Z.; Erts, D.; Smyntyna, V.;

Miele, P. Tuning optical properties of Al2O3/ZnO nanolaminates synthesized by atomic layer deposition. J. Phys. Chem. C 2014, 118, 3811–3819. [CrossRef] 11. Wang, X.O.; Ding, J.N.; Yuan, N.Y.; Cheng, G.G.; Zhu, Y.Y.; Kan, B. Mechanical property of nanoscale

ZnO/Al2O3 multilayers: An investigation by nano-indentation. In Proceedings of the 8th Annual IEEE International Conference on Nano/Micro Engineered and Molecular Systems, Suzhou, China, 7–10 April 2013; pp. 1222–1225. [CrossRef]

12. Guchhait, S.K.; Paul, S. Synthesis and characterization of ZnO-Al2O3 oxides as energetic electro-catalytic material for glucose fuel cell. J. Fuel Chem. Technol. 2015, 43, 1004–1010. [CrossRef] 13. Li, Z.; Yang, R.; Yu, M.; Bai, F.; Li, C.; Wang, Z.L. Cellular Level Biocompatibility and Biosafety of ZnO Nanowires. J. Phys. Chem. C 2008, 112, 20114–20117. [CrossRef] Crystals 2020, 10, 620 15 of 16

14. Rogé, V.; Georgantzopoulou, A.; Mehennaoui, K.; Fechete, I.; Garin, F.; Dinia, A.; Gutleb, A.C.; Lenoble, D. Tailoring the optical properties of ZnO nano-layers and their eﬀect on in vitro biocompatibility. RSC Adv. 2015, 5, 97635–97647. [CrossRef] 15. Oprea, O.; Andronescu, E.; Ficai, D.; Ficai, A.; Oktar, F.; Yetmez, M. ZnO Applications and Challenges. Curr. Org. Chem. 2014, 18, 192–203. [CrossRef] 16. Kołodziejczak-Radzimska, A.; Jesionowski, T. Zinc oxide—from synthesis to application: A review. Materials 2014, 7, 2833–2881. [CrossRef][PubMed] 17. Zhang, W.C.Y.; Nayak, T.R.; Hong, H. Biomedical Applications of Zinc Oxide Nanomaterials. Curr. Mol. Med. 2013, 13, 1633–1645. [CrossRef] 18. Özgür, Ü.; Alivov, Y.I.; Liu, C.; Teke, A.; Reshchikov, M.A.; Dogan, S.; Avrutin, V.; Cho, S.-J.; Morkoç, H. A comprehensive review of ZnO materials and devices. J. Appl. Phys. 2005, 98, 1–103. [CrossRef] 19. Nevarez-Rascon, A.; González-Lopez, S.; Acosta-Torres, L.S.; Nevarez-Rascon, M.M.; Borunda, E.O. Synthesis,

biocompatibility and mechanical properties of ZrO2-Al2O3 ceramics composites. Dent. Mater. J. 2016, 35, 392–398. [CrossRef] 20. George, S.M. Atomic Layer Deposition: An Overview. Chem. Rev. 2010, 110, 111–131. [CrossRef] 21. Kukli, K.; Salmi, E.; Jõgiaas, T.; Zabels, R.; Schuisky, M.; Westlinder, J.; Mizohata, K.; Ritala, M.; Leskelä, M. Atomic layer deposition of aluminum oxide on modiﬁed steel substrates. Surf. Coat. Technol. 2016, 304, 1–8. [CrossRef] 22. Leskelä, M.; Ritala, M. Atomic layer deposition chemistry: Recent developments and future challenges. Angew. Chem. Int. Ed. 2003, 42, 5548–5554. [CrossRef][PubMed] 23. Finch, D.S.; Oreskovic, T.; Ramadurai, K.; Herrmann, C.F.; George, S.M.; Mahajan, R.L. Biocompatibility of atomic layer-deposited alumina thin ﬁlms. J. Biomed. Mater. Res. Part A 2008, 87, 100–106. [CrossRef]

24. Marin, E.; Guzman, L.; Lanzutti, A.; Ensinger, W.; Fedrizzi, L. Multilayer Al2O3/TiO2 Atomic Layer Deposition coatings for the corrosion protection of stainless steel. Thin Solid Films 2012, 522, 283–288. [CrossRef] 25. Beche, E.; Fournel, F.; Larrey, V.; Rieutord, F.; Morales, C.; Charvet, A.-M.; Madeira, F.; Audoit, G.; Fabbri, J.-M.

Direct Bonding Mechanism of ALD-Al2O3 Thin Films. ECS J. Solid State Sci. Technol. 2015, 4, P171–P175. [CrossRef] 26. Nguyen, H.M.T.; Tang, H.-Y.; Huang, W.-F.; Lin, M.C. Mechanisms for reactions of trimethylaluminum with molecular oxygen and water. Comput. Theor. Chem. 2014, 1035, 39–43. [CrossRef] 27. Martínez, E.G.; Flores, J.A.; Tijerina, E.P. Depósito de películas ultradelgadas de óxido de zinc (ZnO) por ALD. CIENCIA-UANL 2010, 13, 247–253. 28. Weckman, T.; Laasonen, K. Atomic layer deposition of zinc oxide: Diethyl zinc reactions and surface saturation from first-principles. J. Phys. Chem. C 2016, 120, 21460–21471. [CrossRef] 29. Guerrero-Sánchez, J.; Borbon-Nunez, H.A.; Tiznado, H.; Takeuchi, N. Understanding the first half-ALD cycle of the ZnO growth on hydroxyl functionalized carbon nanotubes. Phys. Chem. Chem. Phys. 2020, 19, accepted. [CrossRef] 30. Science, R.A. Cytotoxicity Detection Kit Plus (LDH). 2016, pp. 1–24. Available online: https:// www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/Bulletin/1/cytodetrobul.pdf (accessed on 6 July 2020). 31. Kaja, S.; Payne, A.J.; Naumchuk, Y.; Koulen, P. Quantification of lactate dehydrogenase for cell viability testing using cell lines and primary cultured astrocytes. Curr. Protoc. Toxicol. 2017, 2017, 1–10. [CrossRef] 32. Al-Gaashani, R.; Radiman, S.; Daud, A.R.; Tabet, N.; Al-Douri, Y. XPS and optical studies of different morphologies of ZnO nanostructures prepared by microwave methods. Ceram. Int. 2013, 39, 2283–2292. [CrossRef] 33. Scardi, P.; Ermrich, M.; Fitch, A.; Huang, E.W.; Jardin, R.; Kuzel, R.; Leineweber, A.; Mendoza Cuevas, A.; Misture, S.T.; Rebuffi, L.; et al. Size–strain separation in diffraction line profile analysis. J. Appl. Crystallogr. 2018, 51, 831–843. [CrossRef][PubMed] 34. Jlassi, M.; Sta, I.; Hajji, M.; Ezzaouia, H. Effect of nickel doping on physical properties of zinc oxide thin films prepared by the spray pyrolysis method. Appl. Surf. Sci. 2014, 301, 216–224. [CrossRef] 35. Benramache, S.; Belahssen, O.; Arif, A.; Guettaf, A. A correlation for crystallite size of undoped ZnO thin film with the band gap energy—Precursor molarity—Substrate temperature. Optik (Stuttg) 2014, 125, 1303–1306. [CrossRef] Crystals 2020, 10, 620 16 of 16

36. Xiao, S.S.; Zhao, L.; Liu, Y.H.; Lian, J.S. Nanocrystalline ZnO ﬁlms prepared by pulsed laser deposition and their abnormal optical properties. Appl. Surf. Sci. 2013, 283, 781–787. [CrossRef] 37. Iatsunskyi, I.; Kempi´nski,M.; Jancelewicz, M.; Zał˛eski,K.; Jurga, S.; Smyntyna, V. Structural and XPS

characterization of ALD Al2O3 coated porous silicon. Vacuum 2015, 113, 52–58. [CrossRef] 38. Reddy, N.; Bera, P.; Reddy, V.R.; Sridhara, N.; Dey, A.; Anandan, C.; Sharma, A.K. XPS study of sputtered alumina thin ﬁlms. Ceram. Int. 2014, 40, 11099–11107. [CrossRef] 39. Djebaili, K.; Mekhalif, Z.; Boumaza, A.; Djelloul, A. XPS, FTIR, EDX, and XRD analysis of Al2O3 scales grown on PM2000 alloy. J. Spectrosc. 2015, 2015, 1–16. [CrossRef] 40. Das, J.; Pradhan, S.K.; Sahu, D.; Mishra, D.K.; Sarangi, S.; Nayak, B.B.; Verma, S.; Roul, B. Micro-Raman and XPS studies of pure ZnO ceramics. Phys. B Condens. Matter 2010, 405, 2492–2497. [CrossRef] 41. Li, D.H.; Zhai, C.H.; Zhou, W.C.; Huang, Q.H.; Wang, L.; Zheng, H.; Chen, L.; Chen, X.; Zhang, R.J. Eﬀects of Bilayer Thickness on the Morphological, Optical, and Electrical Properties of Al2O3/ZnO Nanolaminates. Nanoscale Res. Lett. 2017, 12.[CrossRef] 42. Martínez-Castelo, J.R.; López, J.; Domínguez, D.; Murillo, E.; Machorro, R.; Borbón-Nuñez, H.A.; Fernandez-Alvarez, I.; Arias, A.; Curiel, M.; Nedev, N.; et al. Structural and electrical characterization of multilayer Al2O3/ZnO nanolaminates grown by atomic layer deposition. Mater. Sci. Semicond. Process. 2017, 71, 290–295. [CrossRef] 43. Randles, J.E.B. Kinetics of rapid electrode reactions. Discuss. Faraday Soc. 2017, 1, 11–19. [CrossRef] 44. Surviliene, S.; Bellozor, S.; Kurtinaitiene, M.; Safonov, V. Protective properties of the chromium–titanium carbonitride composite coatings. Surf. Coat. Technol. 2004, 176, 193–201. [CrossRef] 45. Stern, M.; Geaby, A.L.; Geary, A. Electrochemical polarization I. A theoretical analysis of the shape of polarization curves. J. Electrochem. Soc. 1957, 104, 56–63. [CrossRef] 46. Inc, T.F.S. Intructions Pierce LDH Cytotoxicity Assay Kit. Thermo. Sci 2014, 0747, 1–7. 47. Provided, M.I. Biological evaluation of medical devices—Part 5: Tests for in vitro cytotoxicity Objectives and uses of AAMI standards and recommended practices. In American National Standard; Standard, A.N., Ed.; Association for the Advancement of Medical Instrumentation: Arlington, VA, USA, 2009; pp. 1–34. ISBN 1570203555.

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).