Silver Linings: Electrochemical Characterization of TiO₂ Sol-Gel Coating on Ti6Al4V with AgNO₃ for Antibacterial Excellence

Abstract

This study aimed to synthesize TiO₂ and silver-containing TiO₂ layers on Ti6Al4V titanium alloy substrates, also known as titanium grade 5 (TiGr5), to provide corrosion resistance and antibacterial activity. The TiO₂ layers were prepared through the sol-gel method and dip-coating technique. Silver introduction into the layers was performed in two different ways. One was the impregnation method by dipping the TiO₂ layer-covered metal in aqueous AgNO₃ solutions of various concentrations (TiO₂/AgNO₃), and the other was by direct introduction of AgNO₃ into the precursor sol (Ag-TiO₂). The two methods for incorporating AgNO₃ into the coating matrix are novel, as they preserve silver in its ionic form rather than reducing it to metallic silver. The samples were put through electrochemical characterization, namely potentiodynamic polarization and electrochemical impedance spectroscopy (EIS), and were tested in Hank’s solution, simulating a physiological environment. The behavior of the layers was monitored over time. Also, the thin layers’ thickness and adhesion to the substrate were determined. Microbiological evaluation of the Ag-doped coatings on glass substrates confirmed their significant bactericidal activity against Gram-negative Escherichia coli. Among the two types of coatings, the impregnated coatings demonstrated the most promising electrochemical performance, as evidenced by both EIS and potentiodynamic polarization analyses.

1. Introduction

A widely used alloy in the medical industry is Ti6Al4V, composed of approximately 88%–91% Ti, 5.5%–6.5% Al, and 3.5%–4.5% V, also known as titanium grade 5 (TiGr5). Because of its compatibility with the human body, it is one of the most frequently used materials in knee and hip prostheses and orthopedic implant fabrications. Despite its excellent resistance toward various corrosive media at medium temperatures, the appearance of corrosion products in implanted people has been reported [1]. Several investigations have been made to determine the amounts of metals transported by biological fluids from the implant surface to the organs. Some reports revealed an increased level of Ti in people having Ti alloy-based prostheses [1]. This conclusion highlights the necessity of preventing the spread of corrosion products in the body by applying protective layers on the surfaces of implants. The most practical solution appears to be the formation of TiO₂ layers. Although thin titania coatings can be produced in many ways, like the microemulsion method, chemical vapor deposition, and chemical precipitation [2], one of the most commonly used methods is the sol-gel one [3,4]. Several techniques [5] are used to apply layers on different substrates, among which spin-coating is frequently used [3,4,6], followed by annealing at different temperatures (between 100° and 500 °C). Dip-coating can be a useful alternative and must be highlighted due to its many advantages: it is suitable for substrates of various shapes, the coating thickness can be adjusted according to the necessity, and it is cost-effective and simple. To provide good wear resistance and improve the mechanical properties, the addition of Au-Ni nanoparticles into the coating matrix was reported, e.g., nanoparticles [7]. By incorporating Cu, the fouling resistance of the titania coating could be enhanced [8].

Another drawback of metallic implants is their limited ability to integrate with the body and their susceptibility to infection, which can be addressed by incorporating therapeutic agents into the coating matrix [9,10]. A key challenge in the therapeutic use of Ti-based alloys as orthopedic implants is the risk of biofilm formation, which can lead to infections. Therefore, providing Ti and its alloys with effective antibacterial properties is crucial. Nanoparticles of gold, copper, silver, magnesium, and palladium are widely used due to their biological activities. Bimetallic nanoparticles are more and more often used for therapeutic purposes, employing their antioxidant, cytotoxic, and anti-fungal properties [11,12,13,14].

Their structure-generated characteristics and surface properties make them suitable for incorporation into coating matrices and use in biomedical applications [15]. Due to its broad-spectrum antibacterial activity, silver (Ag) is a perfect candidate as an antibacterial agent to be applied to implants [16]. This is accomplished in several ways, like the preparation of Ag-incorporated porous TiO₂ obtained with magnetron sputtering and micro-arc oxidation [17], Ti/Ag alloy obtained by spark plasma sintering [18], Ag₂O nanoparticle-doped TiO₂ coatings [19], etc. Silver nanoparticles can be obtained via green synthesis methods, involving plant extracts [20]. It was reported that silver can be introduced into titania coatings by adding it into the precursor sol or by impregnation of mesoporous titania coatings in silver-containing solutions [21]. In the aforementioned report, the pore system of titania was created using a template; however, another report indicated that titania possesses significant intrinsic porosity [22]. It has also been reported [21] that the antibacterial behavior of the coating depends on the characteristics of the Ag content; specifically, activity against E. coli has been attributed to Ag crystalline particles smaller than 50 nm. Additionally, if silver is present in the coating in its ionic state, an antibacterial effect results from its release. This could be useful in practical applications; for example, releasing bioactive molecules from a coated metallic implant may help prevent post-operative complications. The selection of coating deposition and the bioactive agent incorporation technique is crucial for achieving a satisfactory coating structure and ensuring strong adhesion between the coating and the implant.

Continuing our studies on TiO₂ coatings on metals [22,23], the present study aimed to evaluate the electrochemical behavior of titania coatings on TiGr5 alloy obtained via the sol-gel method and dip-coating technique. Silver was introduced into the coating material in two different ways. One was direct introduction of AgNO₃ into the precursor sol and the obtained thin film was annealed (Ag-TiO₂). Alternatively, AgNO₃ was introduced into the coating after the deposited TiO₂ layer on the metal was annealed through simple immersion in the silver-containing solution. This raised the question of how much the metallic substrate’s anti-corrosion and other properties are influenced by the coating method, particularly the introduction of silver. We are looking for the answer to this in the following.

2. Materials and Methods

2.1. Preparation of Physiological Solution

The simulated physiological solution chosen for this study was the widely known Hank’s Balanced Salt Solution, which, due to its buffering system, is highly effective in balancing the pH and osmotic pressure in the medium, making it an appropriate choice for the present investigation. Hank’s simulated physiological solution was prepared by the addition of 8 g/L sodium chloride (NaCl, Merck, Darmstadt, Germany), 0.14 g/L calcium chloride (CaCl₂, Sigma-Aldrich, Buchs, Switzerland), 0.4 g/L potassium chloride (KCl, Fisher Scientific, Loughborough, UK), 0.35 g/L sodium bicarbonate (NaHCO₃, Avantor, Gliwice, Poland), 1.1 g/L C₆H₁₂O₆·H₂O D-glucose monohydrate (C₆H₁₂O₆·H₂O, Carl Roth GmbH, Karlsruhe, Germany), 0.115 g/L sodium dihydrogen phosphate monohydrate (NaH₂PO₄·H₂O, Honeywell Fluka, Seelze, Germany), 0.1 g/L magnesium chloride hexahydrate (MgCl₂·6H₂O, Acros Organics, Geel, Belgium), 0.06 g/L disodium hydrogen phosphate dihydrate (Na₂HPO₄·2H₂O, Alfa Aesar, Kandel, Germany), and 0.06 g/L magnesium sulfate heptahydrate (MgSO₄·7H₂O, VWR International, Leuven, Belgium) to distilled water [24].

2.2. Substrate Pretreatment Process

The TiGr5 substrates were first polished with varying grit-sized abrasive paper (a gradient from roughest to smoothest emery paper) while making sure that between each step of the preparation of the surfaces, the latter were thoroughly washed with distilled water, respectively, and at the end of the polishing steps, an ultrasonic cleaning procedure in acetone (CH₃COCH₃, Fisher Scientific, Loughborough, UK) and ethanol (C₂H₅OH, Merck, Darmstadt, Germany) for 10 min each was implemented in order to remove any lingering residue.

As for the glass slides coated in order to perform the antimicrobial analysis, these were cleaned with distilled water (Milli-Q, Millipore, Molsheim, France), aqueous detergent solution, aqueous 10% w/w sulphuric acid solution (H₂SO₄, Honeywell Fluka, Seelze, Germany), and 2-propanol (VWR International, Leuven, Belgium) to ensure a highly adherent and clean surface.

2.3. Preparation of TiO₂ Sols and Coatings

Thin TiO₂ films were prepared using the sol-gel method and deposited on TiGr5 and glass substrates through the dip-coating technique. Titanium(IV) n-butoxide (Ti[O(CH₂)₃CH₃]₄, Merck, Darmstadt, Germany) was used as the titanium precursor. An amount of 2.5 mL of TNB was dissolved in 11.5 mL of ethanol in a glass beaker at room temperature under continuous stirring. The pH of the solution was adjusted to 1.5 by adding 0.18 mL of nitric acid (HNO₃, Honeywell Fluka, Seelze, Germany). The solution was stirred for 2 h at 60 °C [25].

For the synthesis of the Ag-TiO₂ sol, the same preparation steps as for the TiO₂ sol were used, but with the addition of silver nitrate (AgNO₃, Merck, Darmstadt, Germany). AgNO₃ was added to the ethanol before assembling the sol at a 10⁻² M concentration.

Several types of coatings were applied to the TiGr5 substrates; in addition to the undoped TiO₂ coatings, layers were also prepared in which silver was introduced by impregnating the TiO₂ coating in aqueous solutions of silver nitrate of different concentrations (10⁻¹ M, 10⁻² M, and 10⁻³ M) for 30 min (TiO₂/AgNO₃ layers). After establishing the optimal concentrations for the impregnations, these concentrations of AgNO₃ were also used for the preparation of TiO₂ sols by introducing them into ethanol before assembling the sol itself (these sols formed the basis of the Ag-TiO₂ layers).

The layers were produced by the dip-coating method, a versatile and highly cost-effective method which allows for a wide range of geometries (such as the case of TiO₂ implants) to be coated and studied [26]. The used TiGr5 (30 mm × 10 mm × 1 mm) and glass (20 mm × 20 mm) substrates were immersed in the precursor sols and withdrawn at a constant speed of 12 cm/min. Thermic treatment at 150 °C was applied after preparing the layers for a period of 60 min.

2.4. Electrochemical Characterization

Electrochemical measurements were carried out using a three-electrode cell, where the working electrode was constructed of TiGr5 alloy. An Ag/AgCl reference electrode (Metrohm, Herisau, Switzerland) saturated with KCl was utilized and positioned in a Luggin capillary before being introduced into the electrochemical cell. The counter electrode was platinum wire (ELECTROCHEM, GmbH, Krefeld, Germany). The electrolyte solution consisted of a simulated biofluid, specifically Hank’s solution. The cell was connected to an AUTOLAB potentiostat, PGSTAT302N (Eco Chemie BV, Utrecht, The Netherlands), which was computer controlled.

The corrosion protection properties of various TiO₂ layers were examined through complementary electrochemical measurement techniques, including open circuit potential (OCP), potentiodynamic polarization, and electrochemical impedance spectroscopy (EIS).

The electrochemical measurements commenced with the determination of the open circuit potential (OCP) over a period of one hour, followed by EIS measurements in the frequency range of 10 mHz to 10 kHz, utilizing a sinusoidal current with an amplitude of 10 mV at OCP. Finally, polarization curves were recorded within the range of OCP ± 200 mV, the latter being intentionally conducted last to avoid damaging the thin film due to high overpotential.

The electrochemical data were extracted and processed using Origin Pro Software version 2018 (OriginLab Corporation, Northampton, MA, USA).

2.5. Antimicrobial Evaluation

Antimicrobial analysis was performed on glass slides using a Gram-negative strain of Escherichia coli. The slides were prepared following the process outlined in Section 2.3. After pre-treating the coated glass samples according to the EUCAST protocol, they were decontaminated by exposure to UV light for 10 min. The samples were then placed in 12-well plates containing nutrient broth medium (VWR Chemicals, VWR International GmbH, Vienna, Austria). The bacterial strain was added to the wells at a confluence of 0.5 McFarland turbidity (10 µL in 1 mL medium) and incubated for 24 h at 35 °C. After incubation, the liquid was transferred to a 96-well plate, and optical density was measured at 600 nm using an EPOC BioTek spectrophotometer (BioTek Instruments, Winooski, VT, USA). The results were compared to an untreated control [23].

2.6. Coating Adhesion Determination

Coating adhesion was determined according to ASTM D3359 [27] (Lattice-Notch or Cross-Cut method), which involves creating a series of perpendicular cuts, forming a lattice, on the coating surface, followed by the application of pressure-sensitive duct tape to the area. This is then ripped off in one swift and determined move. The amount of detached coating is then assessed in accordance with international standards.

2.7. Coating Thickness Evaluation

The thickness of the prepared layers was measured with a TROTEC BB25 instrument (Trotec GmbH, Heinsberg, Germany) by placing the device perpendicularly onto the coating surface and lightly pushing down on the pin head that measured the thickness. The device operated on an electromagnetic induction basis by generating a magnetic field that interacted with the coating and changed its inductance. This change in inductance was then used to determine the coating thickness automatically.

3. Results and Discussions

3.1. Electrochemical Evaluation

3.1.1. Electrochemical Impedance Spectroscopy

To gain valuable information about the corrosion process, the obtained systems were thoroughly studied and compared by electrochemical impedance spectroscopy (EIS). In the very first step, the optimal concentration of AgNO₃ was investigated by impregnating the plain TiO₂ coating with three different concentrations (10⁻¹, 10⁻², 10⁻³ M) of AgNO₃ aqueous solution, as previously mentioned. The recorded Nyquist and Bode spectra of these coatings can be seen in Figure 1.

Although the Bode absolute impedance diagram did not reveal significant distinctions among similar coatings, the Nyquist diagram highlighted variations in performance that were not evident in the Bode analysis. It is a well-known fact that TiO₂ coatings, as well as titanium substrates, do not present perfect capacitive loops on EIS, which can be due to non-ideal surface roughness or non-homogenous charge distribution on the surface of the TiO₂ layer as different regions of the coating can exhibit different charge transfer activities [28]. As expected, the bare substrate presented the lowest impedance values compared to the coated substrates. The TiO₂ coating enhanced the sample’s resistance toward polarization. In choosing an optimal concentration for AgNO₃, we could observe that the 10⁻² M concentration exhibited the highest impedance value. For this reason, the 10⁻² M concentration was the optimal concentration later introduced into the sol as well (Ag-TiO₂)

Long-term EIS measurements were carried out on the coated TiGr5 substrates. The comparative measurements were effectuated on the plain TiO₂ coating (Figure 2A), Ag-TiO₂ (Figure 2B), and TiO₂/AgNO₃ (Figure 2C). Over time, Nyquist plots, especially those containing AgNO₃, can reflect complex electrochemical processes occurring at the metal/coating and coating/electrolyte interfaces. The plain (Figure 2A) and impregnated (Figure 2B) coatings showed the highest absolute Z values along the course of the 22-day study, while the coatings containing AgNO₃ incorporated into the coating matrix (Figure 2B) showed the lowest values. The TiO₂/AgNO₃ coatings showed a decrease over time, most likely due to the sustained release of AgNO₃ from the coating, which was an expected phenomenon in the case of impregnated coatings. It is also important to mention that the Ag-TiO₂ coatings showed an initial increase in the first two days, followed by a steady decline, which never went lower than the initial coating values. This could occur for several reasons, one being that a phase of passivation might have occurred after the initial exposure, acting as a stabilizer of the coating system and further acting as a barrier to charge transfer, effectively enhancing the corrosion resistance of the substrate. The passivation process was likely attributed to the presence of AgNO₃ in the coating matrix. This created an active interface where the negative charge of oxygen could shift onto the metal when reaching a certain electrode potential. As a result, the adsorption complexes blocked the active sites of the electrode (substrate), promoting passivation [29]. Furthermore, AgNO₃ in the TiO₂ coating could be reduced to form metallic silver on the metal/coating interface, and this could effectively alter the electrochemical behavior and characteristics of the coating. Additionally, given that TiO₂ coatings are commonly known for being thin, irregular, and porous [30], the chances of local corrosion developing increased dramatically, which could explain the lower values of the discussed coating type [31].

In Figure 3, we can observe the Bode plots of the aforementioned coating systems. All three plots present a capacitive behavior deducted from the shape of the curves. It can be observed that Figure 3A,B do not show significant differences, while C presents a slightly different slope variation over time due to the mobility of the impregnated AgNO₃ (TiGr5/TiO₂/AgNO₃), which was higher than that of AgNO₃ incorporated into the coating (Ag-TiO₂).

The |Z|_0.01Hz value reflects the resistance toward polarization of the different samples. Summarizing the time course of |Z|_0.01Hz for the different samples (Figure 4), one can conclude that the unimpregnated sample experienced the most significant change, while the TiGr5/Ag-TiO₂ sample remained the most stable, despite having the lowest overall performance. The fluctuations in the values might have been correlated with the mobility of Ag⁺ in and out of the coatings. Even so, regardless of the fluctuating values, the presence of AgNO₃ exhibited a constantly higher anti-corrosion resistance.

3.1.2. Polarization Curves

The corrosion rates and the susceptibility of coatings to degradation are most commonly studied with potentiodynamic polarization (PDP) and its Tafel interpretation. These are essentially the graphical equivalents of the relationship between the electrode potential and the current density during a simulated electrochemical reaction. The bare, TiO₂, Ag-TiO₂, and TiO₂/AgNO₃-coated TiGr5 polarization curves were recorded and studied to further explore their corrosion behavior (Figure 5).

The corrosion potentials and corrosion current density values evaluated by Tafel interpretation of the polarization curves are listed in

Table 1. Potential and current density values of potentiodynamic polarization curves by Tafel interpretation.

Sample	E (V)	108 × icorr (µA/cm2)	bc V/dec	ba V/dec
TiGr5	−0.379	1.32	0.082	0.068
TiGr5/TiO2	0.083	0.32	0.152	0.126
TiGr5/Ag-TiO2 10−2 M	0.295	4.25	0.079	0.068
TiGr5/TiO2/AgNO3 10−1 M	0.078	1.99	0.095	0.058
TiGr5/TiO2/AgNO3 10−2 M	0.203	1.17	0.119	0.245
TiGr5/TiO2/AgNO3 10−3 M	0.081	0.977	0.185	0.313

. It can be observed from Table 1 that the plain TiGr5 substrate exhibited higher corrosion current (i_corr) values compared to TiGr5/TiO₂, which proved that, as expected, the presence of the coating reduced the current density and thus the corrosion rate. Added to the reduced corrosion rate, the positive potential shift, which could be observed in both plain TiO₂ and the AgNO₃-doped and impregnated coatings, could be attributed to the formation of a more passive surface, which was responsible for the positive shift of the potential. The fact that the corrosion current density of plain TiO₂ was lower than that of Ag-TiO₂ led to the previously speculated conclusion, which was that the presence of AgNO₃ might have caused local corrosion to occur, which means that, in terms of corrosion protection, the addition of AgNO₃ into the coating matrix was not a favorable step, but it did not significantly affect the substrate itself, only the coating, by promoting delamination due to the accumulation of corrosion products at the metal/coating interface [32]. It should be mentioned that even if AgNO₃ did not improve the anti-corrosion properties of the plain TiO₂ coating, it did not affect it negatively, while also adding stability to the electrochemical system at hand.

By comparing the coatings doped with silver in two different ways, it could be concluded that the corrosion current density of the impregnated TiGr5/TiO₂/AgNO₃ samples was lower.

3.2. Coating Thickness

Determination of the thickness of the prepared layers is an essential part of the electrochemical characterization of the phenomena that occur, but it is also necessary to correctly interpret the obtained results. As expected, the impregnated coatings resulted in higher thickness values, which were attributed to the adsorption of AgNO₃ on the TiO₂ film. The coating thickness values can be seen in

Table 2. Coating thickness values for prepared thin films for TiGr5/TiO₂, TiGr5/Ag-TiO₂, and TiGr5/TiO₂/AgNO₃.

Sample	Coating Thickness (µm)
TiGr5/TiO2	96
TiGr5/Ag-TiO2	96
TiGr5/TiO2/AgNO3	122

.

3.3. Coating Adhesion

Inappropriate coating adhesion can affect the performance and durability, as well as the structural integrity and aesthetics, and most importantly, the corrosion resistance and all-around electrochemical behavior of a coating and, as such, its determination is a crucial step in the overall study. Adhesion was determined by the lattice-notch method and classified by the international ASTM D3359 standard for the cross-cut method [33]. Adherence measurements were taken on freshly coated samples before any electrochemical measurements were conducted. All coatings presented with a 99.9% adhesion rate, which placed them in the 4B adhesion class according to the ASTM classification.

3.4. Antimicrobial Analysis

The antimicrobial action of the produced coatings was also investigated. Even though, from an electrochemical standpoint, only one impregnated concentration was further investigated, the antibacterial effect was studied for all impregnated concentrations, including bare TiO₂ and Ag-TiO₂. The investigation was conducted with the use of an Escherichia coli strain, which is a bacterium most commonly found in human and animal intestines and which causes prevalent foodborne illnesses. Previous studies [34,35] have also tested E. coli against AgNO_3- containing systems, making it suitable for the current purpose as well.

The results of the microbiological tests effectuated on the coated glass substrates can be found in Figure 6, where the coated samples on glass substrates were measured against an untreated sample immersed in a bacterial medium. The higher the absorbance, the higher the strain spread [28], as can be seen in the case of the untreated glass substrate 0 as well as the TiO₂-coated substrate 1. It is worth noting that both impregnated coatings and Ag-TiO₂ coatings demonstrated excellent antibacterial properties. However, it is important to consider that while impregnated coatings may initially have exhibited a stronger antimicrobial effect, this effect faded more quickly due to the dissolution of AgNO₃ in the physiological medium. On the other hand, the incorporated AgNO₃ remained within the coating matrix, providing a slower and continuous antimicrobial effect.

4. Conclusions

A complex system of TiO₂ coatings doped and impregnated with AgNO₃ was produced on TiGr5. Firstly, the optimal concentration for AgNO₃ was determined by the electrochemical evaluation of three (10⁻¹, 10⁻², 10⁻³ M) impregnated concentrations of AgNO₃ into the TiO₂ coating matrix. Examination of the comparative Nyquist spectra resulted in 10⁻² M being the optimal concentration, which was used for further investigations.

Long-term wet electrochemical measurements were carried out, consisting of a series of measurements over the course of 22 days. The plain TiO₂ coating was compared with the AgNO₃-doped and the impregnated TiO₂ coatings. It was concluded that the impregnated (TiO₂/AgNO₃) coating showed the best results, as the doped coatings presented with lower values.

Polarization curves were recorded to further corroborate the conclusions drawn during the EIS investigation. The corrosion current densities and positive corrosion potentials verified the superiority of the impregnated coatings compared to the bare and doped coatings.

Adhesion percentiles and coating thicknesses were determined to back up the electrochemical evaluation.

The antimicrobial tests conducted against the E. coli strain demonstrated a significantly heightened antimicrobial effect in both doped and impregnated coating systems, irrespective of concentration. In brief, the impregnated coatings offered the best results for short-term, intense antimicrobial effects, whereas the doped coatings provided sustainable and continuous antimicrobial support. Both are suitable for applications in biomedicine, with potential for further studies.