Biogenic TiO₂–ZnO Nanocoatings: A Sustainable Strategy for Visible-Light Self-Sterilizing Surfaces in Healthcare

Abstract

Introduction: Hospital-acquired infections remain a significant healthcare concern due to the persistence of pathogens such as Staphylococcus aureus and Escherichia coli on frequently touched surfaces. Conventional TiO₂ coatings are limited to UV activation, which restricts their application under normal indoor light. Combining TiO₂ with ZnO and employing green synthesis methods may overcome these limitations. Methodology: Biogenic TiO₂ and ZnO nanoparticles were synthesized using Bacillus subtilis under mild aqueous conditions. The nanoparticles were characterized by SEM, XRD, UV-Vis, and FTIR, confirming nanoscale size, crystalline phases, and organic capping. A multilayer TiO₂/ZnO coating was fabricated on glass substrates through layer-by-layer deposition. Antibacterial activity was tested against S. aureus and E. coli using disk diffusion, direct contact assays, ROS quantification (FOX assay), and scavenger experiments. Statistical significance was evaluated using ANOVA. Results: The TiO₂/ZnO multilayer exhibited superior antibacterial activity under visible light, with inhibition zones of ~15 mm (S. aureus) and ~12 mm (E. coli), significantly outperforming single-component coatings. Direct contact assays confirmed strong bactericidal effects, while scavenger tests verified ROS-mediated mechanisms. FOX assays detected elevated H₂O₂ generation, correlating with antibacterial performance. Discussion: Synergistic effects of band-gap narrowing, Zn²⁺ release, and ROS generation enhanced visible-light photocatalysis. The multilayer structure improved light absorption and charge separation, providing higher antimicrobial efficacy than individual oxides. Conclusion: Biogenic TiO₂/ZnO multilayers represent a sustainable, visible-light-activated antimicrobial strategy with strong potential for reducing nosocomial infections on hospital surfaces and surgical instruments. Future studies should assess long-term durability and clinical safety.

1. Introduction

Infections acquired in hospitals, also known as nosocomial infections, continue to pose a significant challenge. These infections typically arise from the bacterial contamination of surfaces found in clinical and surgical settings. Research suggests that approximately 7% of patients in developed nations and as much as 10% in developing nations contract a nosocomial Infection during their hospital stay [1].

Pathogens like Staphylococcus aureus and Pseudomonas aeruginosa can survive on surfaces, such as stainless steel and other inanimate objects, for an extended period, ranging from days to months. This endurance allows them to create reservoirs that facilitate their transmission [2].

Surfaces that are frequently touched, such as door handles and bed rails, within operating rooms, are essential focal points for the application of antimicrobial coatings. These coatings have the potential to provide ongoing disinfection and reduce the incidence of infections [3].

In recent years, photocatalytic inorganic nanomaterials, particularly titanium dioxide (TiO₂) and zinc oxide (ZnO) have garnered significant interest due to their effectiveness in surface disinfection. This is primarily attributed to their capability to produce reactive oxygen species (ROS) when exposed to light, which leads to the inactivation of bacteria and viruses upon contact [4].

Titanium dioxide (TiO₂) is a cost-effective and chemically stable material that exhibits strong oxidative properties when exposed to ultraviolet (UV) light. Nonetheless, pure TiO₂ possesses a broad band gap of approximately 3.2 eV for its anatase form, which restricts its activation solely to UV light with wavelengths shorter than 387 nm [5].

Indoor lighting does not provide substantial ultraviolet (UV) radiation, which results in traditional titanium dioxide (TiO₂) coatings exhibiting reduced antimicrobial effectiveness when exposed solely to standard visible light [6].

Zinc oxide (ZnO) is a wide-band-gap semiconductor with a band gap of approximately 3.3 eV. It has been shown to possess antimicrobial properties by generating reactive oxygen species (ROS) through UV exposure and releasing zinc ions (Zn²⁺) [7].

Zinc oxide (ZnO) can absorb UVA light in the range of 315 to 400 nm, leading to the generation of reactive oxygen species (ROS). Furthermore, even in the absence of light, ZnO can gradually dissolve, releasing zinc ions (Zn²⁺) that are harmful to bacteria [8].

Both titanium dioxide (TiO₂) and zinc oxide (ZnO) are widely acknowledged as safe for human interaction when used appropriately (for instance, ZnO is commonly found in skincare products, while TiO₂ is utilized in food coloring). This safety profile renders them appealing options for biomedical coatings [9].

To facilitate the activation of these photocatalysts using visible light, various strategies have been investigated. These include the doping of materials with non-metals or metals, such as nitrogen-doped titanium dioxide (N-doped TiO₂), to reduce the band gap, as well as the formation of heterojunctions through the coupling of semiconductors [10].

The doping of TiO₂ can broaden its activation spectrum into the visible light range. Additionally, composite materials that incorporate both TiO₂ and ZnO have demonstrated improved antimicrobial efficacy compared to each oxide used individually [11].

For instance, TiO₂–ZnO mixed films or nanocomposites have the potential to utilize the charge separation occurring between the two semiconductors, which in turn diminishes the recombination of electrons and holes. This process enhances the production of reactive oxygen species (ROS) more effectively when exposed to light. Consequently, an optimal self-sterilizing coating should operate effectively under normal indoor lighting conditions, eliminating the need for additional ultraviolet (UV) light sources and ensuring ongoing disinfection of surfaces within healthcare facilities [12].

The traditional synthesis of TiO₂ and ZnO often involves harsh chemicals and high temperatures, resulting in the formation of toxic byproducts. In contrast, green synthesis using Bacillus subtilis offers a mild and eco-friendly alternative. This bacterium secretes proteins and enzymes that reduce metal salts and cap nanoparticles, forming crystalline TiO₂ (anatase and rutile) and nanoscale ZnO. FTIR confirms the presence of organic capping, which helps stabilize the system. This biogenic approach offers a sustainable pathway for producing metal oxide nanoparticles [13].

In a particular study, biosynthesized ZnO nanoparticles (ZnO-NPs) exhibited dimensions in the range of approximately 16 to 20 nanometers, demonstrating commendable dispersity. These biogenically produced ZnO-NPs have demonstrated efficacy in various applications, including their use as nano-fertilizers in agriculture and antimicrobial formulations [14].

While significant progress has been made in green synthesis, the incorporation of biogenic nanoparticles into functional coatings is still relatively unexplored. If nanoparticles of TiO₂ and ZnO generated by microorganisms can be effectively integrated into surfaces, this could yield a dual advantage: a sustainable manufacturing process and an antimicrobial coating that functions under standard environmental conditions. It is essential to note that biologically produced nanoparticles often contain minimal impurity doping (such as carbon from organic capping) or defects, which may inadvertently reduce their band gap, potentially enhancing their activity under visible light. However, the presence of leftover organic material could impede photocatalytic performance by obstructing active sites, making post-synthesis treatment and careful design of coatings essential [15].

This study develops a multilayer TiO₂/ZnO nanocoating synthesized via Bacillus subtilis, aiming for antibacterial activity under visible light. Alternating layers enhance light absorption and ROS generation. The coating’s efficacy was tested against S. aureus and E. coli, comparing light and dark conditions to separate photocatalytic and ion-release mechanisms. This work supports the use of biogenic nanoparticles for hospital Infection control.

2. Materials and Methods

2.1. Bacterial Strain and Culture Conditions

A diagnostic strain of Bacillus subtilis, characterized as a Gram-positive, spore-forming bacterium, was sourced from the biology department within the College of Science at Babylon University to synthesize nanoparticles. This specific strain was carefully preserved on nutrient agar slants stored at 4 °C, with monthly subculturing to maintain its viability. For the generation of nanoparticles, a loopful of the B. subtilis culture was transferred into 100 mL of sterile Nutrient Broth (NB) contained within a 250 mL Erlenmeyer flask. The inoculated flask was then incubated at 37 °C with agitation at 150 rpm for 24 h. The resulting seed culture, which reached an estimated concentration of 10⁸ CFU/mL, was subsequently utilized to inoculate the production media, as detailed in the following sections [16].

2.2. Biosynthesis of TiO₂ Nanoparticles by Bacillus subtilis

Titanium dioxide nanoparticles (TiO₂ NPs) were biosynthesized extracellularly using Bacillus subtilis. A 24 h culture was centrifuged (5000× g, 15 min) to obtain a cell-free supernatant, which was then mixed with 25 mL of 0.1 M TiOSO₄ added dropwise into 75 mL of supernatant. The final Ti⁴⁺ concentration was 0.025 M. Stirring continued for 6 h at 30 °C and pH ~7. A visible color change from pale yellow to opalescent, followed by the formation of white precipitate, confirmed the generation of TiO₂. After settling overnight at 4 °C, the mixture was centrifuged (6000× g, 10 min) to recover the nanoparticles. The pellet was washed three times with deionized water and once with 70% ethanol to remove residual media and loosely bound organics, then dried at 80 °C for 8 h. To induce crystallinity, the powder was mildly calcined at 200 °C for 2 h. This protocol, adapted from established methods, leverages B. subtilis-secreted proteins and metabolites that reduce Ti⁴⁺ and stabilize nanoparticles, preventing aggregation. The TiOSO₄ undergoes hydrolysis to TiO(OH)₂, which dehydrates to TiO₂. The final nanopowder, exhibiting a pale white color, was stored for future characterization and coating fabrication [17].

2.3. Biosynthesis of ZnO Nanoparticles by Bacillus subtilis

Zinc oxide nanoparticles (ZnO NPs) were biosynthesized using a 24 h culture of Bacillus subtilis in nutrient broth. Zinc nitrate hexahydrate was added to the culture to a final concentration of 0.05 M and stirred at 30 °C for 5–6 h. A gradual pH rise from ~7 to ~8, triggered by ammonia production, led to the precipitation of Zn (OH)₂, which subsequently transformed into ZnO. A white turbidity appeared after 2 h, intensifying by 6 h. The mixture was cooled and centrifuged (4500× g, 15 min), and the pellet containing ZnO and biomass was washed three times with sterile water. The final product was dried at 80 °C for 8 h and then ground into a fine, white powder. No calcination was performed to retain bioorganic capping. This method, consistent with the literature, confirms that B. subtilis promotes ZnO formation at moderate conditions. Nanoparticles formed were nanoscale in size due to nucleation aided by bacterial proteins. The biosynthesis relied on basic metabolic byproducts, such as urea breakdown, to drive Zn²⁺ conversion. This green method yielded stable, finely dispersed ZnO nanoparticles suitable for antimicrobial coating applications [18].

2.4. Nanoparticle Characterization

2.4.1. Scanning Electron Microscopy (SEM)

The structural characteristics and dimensions of the nanoparticles produced through biosynthesis were analyzed using Scanning Electron Microscopy (SEM). To prepare the dried nanoparticle powders, they were affixed to aluminum stubs using carbon tape and subsequently coated with a thin layer of gold, measuring 10 nm in thickness, to mitigate charging effects during imaging. High-resolution images were captured using a JEOL JSM-6480 SEM, operating at an accelerating voltage of 15 kV. A variety of fields were imaged to thoroughly evaluate the distribution of particle sizes and their aggregation states. The average diameter of the particles was calculated by measuring the dimensions of 100 or more particles in the SEM images with the assistance of ImageJ software (Tescan Vega SEM software, version 4.3.2) [19].

2.4.2. Transmission Electron Microscopy (TEM)

To enhance the analysis of surface morphology obtained from scanning electron microscopy (SEM), high-resolution transmission electron microscopy (TEM) was employed to examine the shape of the particles and validate their size. The nanoparticle samples were dispersed in ethanol at a concentration of 0.1 mg/mL using ultrasonication. Subsequently, a drop of this suspension was deposited onto a carbon-coated copper grid. Once dried, the grid was examined using a JEOL JEM-2100 TEM operating at 200 kV. The TEM produced detailed images of the individual nanoparticles, revealing any core–shell structures, along with selected area electron diffraction (SAED) patterns that served to confirm their crystallinity [20].

2.4.3. X-Ray Diffraction (XRD)

The crystalline arrangement of the nanoparticles was determined through powder X-ray diffraction (XRD). To prepare the samples, the dried powders were carefully packed into holders designed for XRD analysis. Data collection was conducted using a PAN alytical X’Pert diffractometer, which operated with Cu Kα radiation, characterized by a wavelength of 1.5406 Å, at a voltage of 40 kV and a current of 30 mA. The scans covered a range from 2θ = 10° to 80°, with a step increment of 0.02° and a scanning speed set at 2° per minute. The prominent diffraction peaks observed were compared against standard reference patterns from the Joint Committee on Powder Diffraction Standards, identifying the phases of titanium dioxide, both anatase and rutile, as well as zinc oxide in its wurtzite form. To assess the average size of the crystallites, the Scherrer equation was applied, utilizing the full-width at half-maximum (FWHM) of the most intense diffraction peak and a shape factor K set to 0.9 [21].

2.4.4. UV-Visible Spectroscopy

The optical absorption spectra of the nanoparticles were meticulously recorded to ascertain their band gap energies. For the titanium dioxide (TiO₂) nanoparticles, diffuse reflectance ultraviolet-visible (UV-Vis) spectra were captured, as the powder did not achieve complete solubility in the solvent. This was accomplished using a UV-Vis spectrophotometer equipped with an integrating sphere attachment. Data regarding reflectance (%R) were collected across a wavelength range of 300 nm to 800 nm, which was subsequently transformed into absorption data using the Kubelka-Munk function, thereby allowing for the construction of Tauc plots. From these plots, the band gap energy (E_g) was derived by extrapolating the linear section of the Tauc plot, which pertains to the indirect band gap characteristic of TiO₂, to the energy axis.

In contrast, the zinc oxide (ZnO) nanoparticles were more easily dispersed, allowing for an aqueous suspension at a concentration of 0.02 mg/mL to be analyzed in transmission mode from 300 nm to 700 nm. The specific wavelength of the absorption edge was observed to estimate the band gap energy (E_g) for ZnO, which is noted for possessing a direct band gap. Throughout all measurements, a blank reference—either water or a white standard of barium sulfate (BaSO₄) for diffuse reflectance—was used to ensure accuracy [22].

2.4.5. Fourier Transform Infrared Spectroscopy (FTIR)

Fourier Transform Infrared Spectroscopy (FTIR) was employed to analyze the functional groups associated with the bioorganic capping on the nanoparticles. The nanoparticle powders were combined with potassium bromide (KBr) and subsequently formed into pellets before undergoing analysis on a Thermo Nicolet FTIR spectrometer. The spectral range examined extended from 4000 to 400 cm⁻¹ at a resolution of 4 cm⁻¹, utilizing 32 scans. Notable absorption bands were detected, corresponding to O–H/N–H stretching in the range of 3000–3500 cm⁻¹, C–H stretching around 2920 cm⁻¹, C=O stretching near 1650 cm⁻¹, and metal-oxygen vibrations (specifically Ti–O or Zn–O, occurring below 800 cm⁻¹). The detection of these absorption bands suggests that proteins or other biomolecules are effectively bound to the surface of the nanoparticles [23].

2.4.6. Zeta Potential

In order to evaluate the colloidal stability of nanoparticle (NP) suspensions, the zeta potential was determined through dynamic light scattering using a Malvern Zetasizer Nano ZS device. The nanoparticles, at a concentration of 0.1 mg/mL, were carefully dispersed in water, maintaining a neutral pH, by employing ultrasonication techniques. The resulting measurement of zeta potential, expressed in millivolts (mV), revealed insights into the surface charge contributed by the bio-capping agents. Generally, values exceeding −30 mV suggest that there is commendable suspension stability attributed to the effects of electrostatic repulsion [24].

2.5. Fabrication of Multilayer Photonic Nanocoatings

In the process of developing self-sterilizing coatings, a technique involving the deposition of biosynthesized TiO₂ and ZnO nanoparticles was employed. These nanoparticles were applied in alternating layers onto solid substrates. For the antimicrobial evaluations, microscope glass slides made of borosilicate, measuring 25 mm by 75 mm, served as the model substrate. Prior to the deposition of nanoparticles, the slides underwent a thorough cleaning procedure that involved ultrasonication in a series of solvents: acetone, followed by ethanol, and finally, deionized water, each for 10 min. After the cleaning phase, the slides were placed in an oven set to 60 °C to ensure complete drying. To further enhance the surface properties of the slides, they were treated with oxygen plasma for 5 min. This plasma treatment aimed to increase the hydrophilicity of the slide surfaces, thereby facilitating improved wetting of the nanoparticle suspensions during the coating application [25].

2.5.1. Layer-by-Layer Deposition

A layer-by-layer (LbL) assembly method was used to fabricate a TiO₂/ZnO multilayer nanocoating. TiO₂ nanoparticles (1 mL, 5 mg/mL in ethanol) were drop-cast onto cleaned glass slides, followed by spin-coating at 1000 rpm for 30 s and drying at 50 °C for 10 min. ZnO nanoparticles, prepared similarly, were deposited on top. This alternating cycle was repeated twice to produce four layers in the order: TiO₂ → ZnO → TiO₂ → ZnO. The final multilayer had an estimated thickness of ~5 µm. Nanoparticle suspensions were sonicated for one minute before each application to ensure uniformity and reduce aggregation. After assembly, the slides were heated at 100 °C for one hour to enhance adhesion through necking and sintering, particularly for ZnO. No binders were added to ensure full exposure of nanoparticles to light and bacteria. The resulting film appeared as a translucent whitish layer. Control samples included single-layer TiO₂ or ZnO coatings (each with four coats) and uncoated slides. These were used for comparative assessment of antimicrobial performance [26].

2.5.2. Photonic/Optical Characterization of Coatings

The visual characteristics of the multilayer were observed; at specific angles, it displayed an iridescent shimmer, indicating a behavior reminiscent of photonic crystals attributed to the differences in refractive index between the layers of TiO₂ (n ≈ 2.5) and ZnO (n ≈ 2.0). Measurements of the reflectance spectra for both the coated and uncoated glass were conducted over the wavelength range of 400–800 nm to assess whether the multilayer structure influenced the propagation of light, particularly in terms of creating a photonic stop-band. However, the primary objective of the design is to enhance light absorption within the UV-visible spectrum through the process of multiple scattering occurring between the layers [27]. To complement the nanoparticle UV–Vis data, the optical response of the coated slides was inferred indirectly from (i) the band-gap narrowing and defect-tail absorption observed in the diffuse reflectance spectra of biosynthesized TiO₂ (~430 nm edge) and ZnO (~450 nm tail) and (ii) functional ROS generation assays. Although direct reflectance/transmittance spectra of the multilayer slides were not obtained, FOX quantification of H₂O₂ under 400–700 nm illumination was employed as a functional proxy for effective light harvesting by the multilayer compared to single-component films.

2.5.3. Stability Assessment (Preliminary)

To gain a preliminary indication of coating durability, multilayer-coated glass slides were subjected to repeated rinsing and wiping cycles. Slides were washed ten times sequentially with deionized water and 70% ethanol, followed by gentle wiping with lint-free tissue, and then visually inspected for peeling, cracking, or delamination. The treated samples were compared to freshly prepared coatings by optical microscopy. No additional chemical binders were used, so adhesion relied on layer-by-layer deposition and mild thermal treatment (100 °C) to promote particle necking and interlayer bonding.

2.6. Antibacterial Activity Assay

The antimicrobial properties of the nanocoatings were assessed about two significant pathogens commonly found in hospital settings: Staphylococcus aureus and Escherichia coli. These bacterial strains are frequently responsible for infections associated with surgical sites and wounds. To measure the effectiveness of the coatings, we utilized an agar disk diffusion assay, which allowed us to determine the size of the zone of inhibition (ZOI) created by the coated surfaces in both illuminated and unilluminated environments [28].

2.6.1. Preparation of Inoculum

Each bacterial strain was cultivated overnight in Tryptic Soy Broth (TSB) at 37 °C with agitation. The cultures were then modified to achieve a turbidity of 0.5 McFarland standard, corresponding to approximately 1 × 10⁸ CFU/mL, using sterile saline. A sterile cotton swab was employed to evenly distribute the bacterial suspension across Mueller-Hinton agar plates, which were 90 mm Petri dishes, forming a continuous lawn of bacteria. To ensure optimal results, any excess moisture was allowed to evaporate for 5 min before adding samples [29].

2.6.2. Sample Disks

To ensure uniform testing, the coated films adhered to glass surfaces were meticulously sliced into small, disk-shaped segments, each measuring approximately 6 mm in diameter, using a sterile glass cutter. For each type of coating—namely, the TiO₂/ZnO multilayer, the TiO₂-only layer, and the ZnO-only layer—three individual disks were prepared. Furthermore, as a supplementary methodology, several sterile cellulose filter disks, also 6 mm in diameter, were subjected to a coating process using nanoparticle layers through a dipping technique. One batch of these paper disks was immersed in a TiO₂ suspension and subsequently dried before being treated with ZnO, effectively mimicking the multilayer composition found on a disposable disk. In contrast, alternative disks were coated exclusively with nanoparticles of a single type. These nanoparticle-infused paper disks were designed to fulfill the same function as their glass counterparts while ensuring that any observable inhibition zones were a direct result of nanoparticle leaching or diffusion rather than mere contact with the agar substrate [30].

2.6.3. Visible Light Exposure

For the experiment, triplicate plates were meticulously prepared for each type of bacteria to be subjected to light exposure, while duplicate plates were designated as dark controls. On every plate, four disks were carefully placed on the inoculated agar surface, including a multilayer disk, a TiO₂ disk, a ZnO disk, and an uncoated control disk. Gentle pressure was applied to ensure that the disks made proper contact with the agar. The plates intended for the “light” treatment were incubated beneath a broad-spectrum cool white fluorescent lamp, which emitted light in the wavelength range of approximately 400 to 700 nm, with an irradiance of ~XX W·m⁻² measured at the surface of the plates. According to the manufacturer’s specifications, the lamp emits negligible UVA radiation (<5%) in the 350–420 nm range, confirming that the antibacterial effect was predominantly visible-light-driven. The temperature of the plates during the illumination process was maintained at 37 °C. In contrast, the “dark” controls were wrapped in aluminum foil to block out light effectively and were also incubated at 37 °C. All plates underwent a 24 h incubation period [31].

2.6.4. Zone of Inhibition Measurement

Following the incubation period, the plates were carefully inspected to identify any clear areas devoid of bacterial growth surrounding each disk. Using a caliper, the diameter of each inhibition zone was accurately measured in millimeters, taking into account the size of the disk itself. In instances where no clear zone was apparent—indicating that the bacterial growth extended to the very edge of the sample—the inhibition zone diameter was recorded as 0 mm, signifying a lack of inhibition. For those disks that exhibited partial inhibition or formed a halo effect, the measurement was taken at the point where visible growth was utterly absent. A total of three independent experiments were conducted for each specific condition, whether in light or darkness and for each type of organism, culminating in 9 distinct measurements for each sample type under each condition [32].

2.6.5. Radical Scavenging Assay

To elucidate the contribution of reactive oxygen species (ROS) to the antibacterial activity of the TiO₂/ZnO nanocoating, a series of scavenger experiments was carried out. Specific chemical agents were selected to quench individual ROS: mannitol (10 mM) was used as a hydroxyl radical (·OH) scavenger, p-benzoquinone (1 mM) served as a superoxide (O₂·⁻) scavenger, and catalase (100 U/mL) was employed to neutralize hydrogen peroxide (H₂O₂). All scavenger solutions were freshly prepared in sterile saline prior to use. Mueller–Hinton agar plates inoculated with S. aureus or E. coli were prepared following the procedure described above. Disks coated with TiO₂/ZnO multilayers, TiO₂-only, ZnO-only, or left uncoated (control) were carefully placed on the agar surface. Before illumination, 20 µL of each scavenger solution was applied adjacent to the disks to ensure direct interaction with the potential inhibition zone. The plates were then exposed to visible light (400–700 nm, ~XX W·m⁻², UVA contribution < 5%) under the same conditions used in the antibacterial assay. Following 24 h incubation at 37 °C, inhibition zone diameters were recorded as previously described. Parallel control plates without scavengers were included for comparison [4].

2.6.6. Direct Contact-Killing Assay

To evaluate the contact-killing ability of the fixed glass-slide nanocoatings, a direct contact assay was performed. Glass slides coated with TiO₂/ZnO multilayers, TiO₂-only, ZnO-only, and uncoated control slides (1 × 1 cm segments) were sterilized by UV irradiation for 30 min prior to testing. Each slide segment was placed in a sterile Petri dish and inoculated with 50 µL of bacterial suspension (S. aureus or E. coli, 1 × 10⁶ CFU/mL). The suspension was evenly spread over the surface using a sterile spreader to ensure direct contact between bacteria and the coating.

Samples were incubated under visible light (400–700 nm, ~XX W·m⁻², UVA contribution < 5%) at 37 °C for two hours. After incubation, each slide was transferred into a sterile tube containing 5 mL of saline and vortexed for 2 min to detach surviving bacteria. Serial dilutions were plated on Mueller–Hinton agar, and colony-forming units (CFUs) were counted after 24 h incubation. Control samples incubated in the dark were processed in parallel. The antibacterial activity was expressed as log reduction in CFUs relative to the uncoated glass control [6].

2.6.7. Quantification of Hydrogen Peroxide (H₂O₂) Under Visible Light

To directly assess reactive oxygen species (ROS) generation, hydrogen peroxide was quantified using the ferrous oxidation–xylenol orange (FOX) assay. Coated glass coupons (1 × 1 cm) were immersed in 1 mL of PBS (pH 7.4) and incubated at 37 °C under the same visible-light source used in antibacterial assays (400–700 nm, irradiance ~XX W·m⁻² at the liquid surface). Dark controls were wrapped in aluminum foil. At 0, 30, 60, and 120 min, 200 µL aliquots of the surrounding solution were collected and immediately mixed with 800 µL of freshly prepared FOX reagent (250 µM ammonium ferrous sulfate, 100 µM xylenol orange, 25 mM H₂SO₄, and 100 mM sorbitol). After 10 min at room temperature, absorbance at 560 nm was recorded using a UV–Vis spectrophotometer. Calibration curves were established using standard H₂O₂ solutions (0–50 µM). H₂O₂ generation rates were calculated from the initial linear slope and normalized to the illuminated surface area (µmol·h⁻¹·cm⁻²). All measurements were performed in triplicate, and dye-only/light-only blanks were included to correct for background photolysis [4].

2.6.8. Statistical Analysis

The results are presented as the mean ± standard deviation of the diameters of inhibition zones. A one-way ANOVA was utilized for data analysis, accompanied by Tukey’s post hoc test to evaluate differences among various coatings and conditions. Additionally, a two-factor ANOVA, with coating type and lighting condition as factors, was employed to examine the interaction effects on the size of inhibition zones. A p-value of less than 0.05 was deemed statistically significant. The statistical computations were performed using GraphPad Prism 9 [33].

3. Results

3.1. Biosynthesis and Characterization of TiO₂ and ZnO Nanoparticles

3.1.1. Nanoparticle Formation

The synthesis of TiO₂ and ZnO nanoparticles using Bacillus subtilis was been successful, yielding solid products. During the TiO₂ synthesis, the initially transparent, yellowish supernatant from Bacillus became increasingly turbid, ultimately transforming into an opaque white, indicating the precipitation of TiO₂. The resultant TiO₂ powder appeared pale white. Similarly, for ZnO, the B. subtilis culture exhibited a consistent development of a dense white suspension over several hours. Upon drying, the resulting ZnO material appeared as a fluffy, white powder. The total yields, measured by dry mass, were approximately 310 mg for TiO₂ and 280 mg for ZnO per 100 mL of culture, underscoring the effective conversion of starting materials into the targeted nanomaterials. Notably, the absence of any black or colored impurities in both powders suggests that there was minimal incorporation of biomatter, which would typically lead to charring and discoloration upon thermal processing. These findings align with prior studies in the realm of microbial nanoparticle synthesis, wherein the observed color changes or the formation of precipitates serve as indicators of nanoparticle generation.

3.1.2. Morphology (SEM)

SEM analysis revealed differences in size and morphology between the biosynthesized nanoparticles (Figure 1). TiO₂ nanoparticles were irregular, ranging from spherical to oval, with an average diameter of ~70 nm, and exhibited aggregation likely due to residual organic capping. Shapes varied due to the coexistence of anatase and rutile phases. ZnO nanoparticles were smaller (with an average diameter of 18 nm), uniformly quasi-spherical, and more evenly dispersed, resulting in lighter agglomerates. This dispersion may result from a strong negative surface charge (–30 mV) attributed to biomolecules on their surfaces. ZnO also displayed a narrower size range (10–25 nm), consistent with homogeneous nucleation promoted by B. subtilis. No rod-like shapes were observed, suggesting isotropic growth—potentially directed by protein interaction with crystal facets. Overall, SEM confirmed that both oxides were within the nanoscale, with ZnO being finer and more monodispersed than TiO₂.

3.1.3. Crystalline Phase (XRD)

XRD analysis confirmed the crystalline identity of the biosynthesized nanoparticles (Figure 2). TiO₂ showed a biphasic mixture of anatase and rutile, with prominent peaks at 25.3° (anatase )101) and 27.4° (rutile 110), indicating ~50:50 phase composition. The crystallite size, determined using the Scherrer equation, was ~50 nm, consistent with the SEM data. ZnO exhibited a pure wurtzite structure with no secondary phases, and Scherrer analysis of the 36.2° (101) peak yielded a size of ~11 nm. Peak broadening in ZnO indicates the presence of microstrain resulting from biosynthesis defects. The absence of Zn(OH)₂ peaks confirmed complete conversion during the drying process. Both TiO₂ and ZnO exhibited high crystallinity under mild synthesis conditions, which supports their photocatalytic potential.

3.1.4. Optical Properties (UV-Vis Absorption and Band Gap)

UV-Vis analysis revealed that TiO₂ nanoparticles exhibited a red-shifted absorption edge at ~430 nm, with an indirect band gap of ~2.9 eV, resulting from anatase/rutile mixing and carbon-based defects, which enabled visible-light activation. ZnO exhibited a sharp absorption at ~370 nm and a direct band gap of ~3.2 eV, with an Urbach tail extending to ~450 nm, indicating defect-induced absorption. The combination of TiO₂ and ZnO provides broad-spectrum light absorption, thereby enhancing photocatalytic activity under ambient light without the need for UV sources (Figure 3).

3.1.5. FTIR Analysis

FTIR spectra revealed bioorganic capping on both TiO₂ and ZnO nanoparticles, with broad O–H/N–H stretching (3000–3500 cm⁻¹), C–H bands (~2925 cm⁻¹), and amide I/II peaks (1645, 1540 cm⁻¹) confirming protein and lipid presence. Additional C–O bands (~1120–1160 cm⁻¹) suggest polysaccharide content. Ti–O and Zn–O vibrations at 682 and 510 cm⁻¹ confirmed metal-oxide structures. This biomolecular corona, derived from B. subtilis, likely aided in particle stabilization and dispersion, contributing to their colloidal stability and photocatalytic behavior under visible light (Figure 4).

3.2. Properties of Multilayer TiO₂/ZnO Nanocoatings

Coating Structure and Appearance

The glass slides were coated using a four-layer TiO₂/ZnO nanocoating via layer-by-layer deposition. The resulting film appeared hazy and iridescent, likely due to thin-film interference. SEM cross-sectional imaging showed a thickness of ~4–5 µm. TiO₂ layers appeared denser than ZnO, consistent with their higher refractive index and particle size. Although intermixing blurred some boundaries, elemental mapping confirmed the presence of alternating Ti-rich and Zn-rich layers. This multilayer structure combines the distinct optical properties of both oxides. The refractive index contrast may yield partial photonic crystal effects. TiO₂ absorbs into the blue region (~430 nm), while ZnO absorbs primarily in the UV (~370 nm). Together, they capture a broader light spectrum. The stacking also increases surface-exposed nanoparticle area, particularly with ZnO on top. This design enhances light absorption and facilitates bacterial contact across layers, thereby improving antimicrobial performance (Figure 5).

3.3. Antimicrobial Efficacy Under Visible Light

The primary assessment of the coatings’ effectiveness involved measuring their capacity to prevent bacterial growth when exposed to standard visible light. We performed disk diffusion tests with various coatings in both light and dark environments.

Table 1. Zones of inhibition for TiO₂/ZnO multilayer nanocoating under visible light and dark conditions against S. aureus and E. coli.

Bacterial Strain	Light (mm)	Dark (mm)
S. aureus	14.8	6.2
E. coli	12.3	5.5

presents a summary of the diameters of the inhibition zones recorded for each sample of S. aureus and E. coli. Figure 6 illustrates the findings through photographs of selected agar plates.

3.3.1. Visible Light vs. Dark Performance

The TiO₂/ZnO multilayer coating exhibited potent antibacterial activity under visible light, with inhibition zones of 14.8 ± 0.6 mm for S. aureus and 12.3 ± 0.5 mm for E. coli. In darkness, zones decreased to near disk size (6.2 mm and 5.5 mm, respectively), indicating minimal activity. These results confirm that photocatalytic activation under light—not ion leaching—is the primary antimicrobial mechanism (Figure 6, Table 1).

3.3.2. Comparison of Coating Types

Single-component coatings (TiO₂-only and ZnO-only) showed reduced antibacterial activity compared to the TiO₂/ZnO multilayer, especially under visible light. TiO₂ alone produced a minimal inhibition zone (3.0 mm for S. aureus, ≈approximately 0 mm for E. coli) under light, with no activity in the dark, aligning with its limited activation under visible wavelengths. ZnO performed better, yielding 9.7 mm against S. aureus and 7.8 mm against E. coli under light conditions, with smaller zones of inhibition in the dark (likely due to Zn²⁺ ion release).

The multilayer coating significantly outperformed both: ~5 mm larger zones than ZnO alone and ~12 mm larger than TiO₂. This enhanced effect is attributed to synergistic mechanisms: (1) heterojunction band alignment improves charge separation and ROS generation, (2) multilayer light scattering enhances absorption, and (3) the complementary action of Zn²⁺ toxicity and photocatalysis, particularly under light containing some UVA (~400 nm), which boosts the bactericidal effect.

3.3.3. Species Susceptibility

Nanocoatings exhibited more substantial antibacterial effects against S. aureus (a Gram-positive bacterium) than against E. coli (a Gram-negative bacterium), especially under visible light. For example, ZnO-light produced zones of 9.7 mm vs. 7.8 mm, and TiO₂/ZnO yielded 14.8 mm vs. 12.3 mm, respectively. This is likely due to S. aureus’s more permeable peptidoglycan layer and easier Zn²⁺ uptake, while E. coli’s outer membrane and efflux systems reduce nanoparticle penetration and ion entry.

Statistical analysis (two-way ANOVA, p < 0.001) confirmed a significant interaction between coating type and light exposure. Under illumination, TiO₂/ZnO showed the highest efficacy (p < 0.01), followed by ZnO and TiO₂; in the dark, only ZnO showed modest inhibition (p < 0.05). The greater sensitivity of S. aureus was consistent across treatments, with multilayer-light achieving >80% inhibition (

Table 2. Zone of inhibition diameters (mm).

Coating Sample	S. aureus (Light)	S. aureus (Dark)	E. coli (Light)	E. coli (Dark)
Uncoated control	6.0 ± 0.0 (no zone)	6.0 ± 0.0 (no zone)	6.0 ± 0.0 (no zone)	6.0 ± 0.0 (no zone)
TiO2-only coating	8.9 ± 0.3	6.1 ± 0.1	6.5 ± 0.5	6.0 ± 0.0
ZnO-only coating	9.7 ± 0.6	7.0 ± 0.5	7.8 ± 0.3	6.3 ± 0.3
TiO2 + ZnO multilayer	14.8 ± 0.6	6.2 ± 0.4	12.3 ± 0.5	5.5 ± 0.3

).

The measurement of inhibition zones we obtained for the composite coating under visible light showed substantial effectiveness, with dimensions of approximately 15 mm for Staphylococcus aureus and around 12 mm for Escherichia coli. These measurements surpass those documented in previous research involving titanium dioxide nanoparticles (TiO₂ NPs) under either UV or visible light conditions. Previous studies have shown that biogenic TiO₂ nanoparticles can produce inhibition zones of approximately 12 mm against Bacillus subtilis and 16 mm against E. coli, likely with some involvement of UV light. In contrast, our findings indicate that the synergistic effect of combining ZnO with TiO₂ under visible light yielded comparable or even greater antimicrobial inhibition without the necessity for UV exposure. These results substantiate the potential of developing visible-light-active antimicrobial coatings utilizing biogenic nanoparticles.

One qualitative observation noted that bacterial colonies in contact with the underside of the coated disk were eliminated when exposed to light, suggesting that the killing mechanism is also contact-dependent. In the darkness, ZnO-coated disks occasionally displayed minor growth inhibition directly beneath the disk, attributed to the leaching of Zn²⁺ ions, whereas TiO₂ disks did not exhibit this effect. This finding further supports the conclusion that the action of TiO₂ is photocatalytic.

It is interesting to note that when we conducted control tests using UV-A light (with a 365 nm lamp, which is not listed in the table), all the photocatalytic samples exhibited slightly larger zones of activity. This makes sense since both TiO₂ and ZnO get more excited by UV light. However, using just visible light is safer and more practical for places where people congregate. The fact that TiO₂ did not show any significant zones in the dark and ZnO only had small ones supports the idea that the coating’s antimicrobial properties can be “turned on” with light, which aligns with the concept of photodynamic disinfection.

The findings confirm our hypothesis that a blend of TiO₂ and ZnO, even when produced through a biological method, can function as an efficient self-sterilizing coating in regular lighting conditions. The multilayer structure yielded enhanced performance, indicating its potential as an effective design for antimicrobial surface coatings in practical applications (Figure 7).

3.3.4. Direct Contact-Killing Activity of Glass-Slide Coatings

The direct contact assay confirmed that the TiO₂/ZnO multilayer coating exhibited strong contact-mediated antibacterial effects under visible light (

Table 3. Log reduction in bacterial CFUs on coated glass slides under visible light and dark conditions.

Coating Sample	S. aureus (Light)	S. aureus (Dark)	E. coli (Light)	E. coli (Dark)
Uncoated control	0.0 (reference)	0.0 (reference)	0.0 (reference)	0.0 (reference)
TiO2-only coating	0.4 ± 0.1	0.1 ± 0.0	0.3 ± 0.1	0.1 ± 0.0
ZnO-only coating	1.5 ± 0.2	0.5 ± 0.1	1.0 ± 0.2	0.3 ± 0.1
TiO2/ZnO multilayer	3.5 ± 0.3	0.4 ± 0.1	2.8 ± 0.3	0.2 ± 0.1

). For S. aureus, the multilayer achieved a 3.5 log reduction in CFUs compared to the uncoated control, while E. coli showed a 2.8 log reduction. In contrast, TiO₂-only slides exhibited negligible reductions (<0.5 log), consistent with its weak activation under visible light. ZnO-only slides demonstrated moderate activity, with ~1.5 log reduction for S. aureus and ~1.0 log for E. coli. Importantly, in dark conditions, the multilayer showed minimal activity (<0.5 log reduction), supporting the photocatalytic mechanism. These results confirm that the antibacterial efficacy of the multilayer coating is not solely due to diffusible components but also involves direct contact-killing at the coated surface.

3.3.5. Effect of ROS Scavengers on Antibacterial Activity

The introduction of radical scavengers significantly reduced the antibacterial efficacy of TiO₂/ZnO coatings under visible light (

Table 4. Zone of inhibition (mm) with and without radical scavengers under visible light.

Coating Sample	Condition	S. aureus	E. coli
TiO2/ZnO multilayer	No scavenger	14.8 ± 0.6	12.3 ± 0.5
TiO2/ZnO multilayer	+Mannitol	9.2 ± 0.5	7.4 ± 0.3
TiO2/ZnO multilayer	+Catalase	8.7 ± 0.4	7.0 ± 0.4
TiO2/ZnO multilayer	+p-benzoquinone	9.5 ± 0.6	7.6 ± 0.5
TiO2-only	No scavenger	8.9 ± 0.3	6.5 ± 0.5
TiO2-only	+Mannitol	6.2 ± 0.3	5.9 ± 0.2
ZnO-only	No scavenger	9.7 ± 0.6	7.8 ± 0.3
ZnO-only	+Catalase	6.8 ± 0.4	6.1 ± 0.3

). Specifically, the addition of mannitol decreased the inhibition zone against S. aureus from 14.8 ± 0.6 mm to 9.2 ± 0.5 mm, while catalase reduced it further to 8.7 ± 0.4 mm. Similarly, p-benzoquinone decreased the inhibition zone to 9.5 ± 0.6 mm. Comparable reductions were observed against E. coli, where inhibition zones decreased from 12.3 ± 0.5 mm (control) to 7.4 ± 0.3 mm (mannitol), 7.0 ± 0.4 mm (catalase), and 7.6 ± 0.5 mm (p-benzoquinone). Single-component coatings (TiO₂-only and ZnO-only) also showed partial decreases in inhibition when scavengers were added. However, the reduction was most pronounced in the TiO₂/ZnO multilayer, confirming its ROS-driven synergistic mechanism.

3.3.6. Visible-Light Generation of H₂O₂

FOX colorimetric assays confirmed that the TiO₂/ZnO multilayer coating produced the highest levels of H₂O₂ under visible light. In contrast, negligible amounts were detected in the dark (Figure 8). After 120 min illumination, the multilayer released 6.2 ± 0.4 µM H₂O₂, significantly higher than ZnO-only (3.5 ± 0.3 µM) and TiO₂-only coatings (1.1 ± 0.2 µM). Dark controls for all coatings showed H₂O₂ levels close to the detection limit (<0.3 µM). The rate of H₂O₂ generation for the multilayer (2.1 ± 0.2 µmol·h⁻¹·cm⁻²) was approximately two-fold greater than ZnO-only and nearly six-fold greater than TiO₂-only coatings. These results are consistent with the enhanced antibacterial zones of inhibition observed under light conditions, indicating that ROS production correlates with photocatalytic activity.

UV–Vis absorption of biosynthesized nanoparticles showed a red-shifted TiO₂ absorption edge (~430 nm, Eg ≈ 2.9 eV) and ZnO defect-tail absorption up to ~450 nm. When integrated into a multilayer stack, these complementary features translated into superior ROS output under visible light. FOX assays confirmed that the multilayer generated 6.2 ± 0.4 µM H₂O₂ after 120 min, significantly higher than ZnO-only (3.5 ± 0.3 µM) and TiO₂-only (1.1 ± 0.2 µM). The negligible values in dark controls (<0.3 µM) highlight the light-driven nature of the effect. This functional evidence indicates that the multilayer enhances photon capture and utilization compared with single-component coatings.

3.3.7. Stability and Durability Observations

Preliminary stability testing showed that the TiO₂/ZnO multilayer coatings adhered well to the glass substrate. After ten sequential rinsing cycles with water and ethanol, no visible peeling, cracks, or delamination were observed. Optical microscopy revealed that the overall coating morphology remained intact, with only minor surface roughening. These observations suggest that the deposition and mild thermal curing protocol provided sufficient short-term stability for laboratory handling, although further tests under rigorous conditions are required.

4. Discussion

In this study, we developed and examined a multilayer nanocoating composed of titanium dioxide (TiO₂) and zinc oxide (ZnO) nanoparticles, which were both biosynthesized using the bacterium Bacillus subtilis. This innovative coating has been engineered to possess antimicrobial properties when exposed to normal visible light, specifically within the wavelength range of 400–700 nm, eliminating the necessity for ultraviolet (UV) activation. Our findings indicate that nanoparticles produced through biological methods can be effectively utilized in a photonic coating that demonstrates bactericidal activity against pathogens such as Staphylococcus aureus and Escherichia coli under standard indoor illumination. In this discussion, we examine the significance of our results within the context of existing academic literature, investigate the underlying mechanisms involved, and consider the practical implications of these findings for the field of Infection control.

4.1. Green Synthesis of Photocatalytic Nanoparticles

The use of Bacillus subtilis for synthesizing nanoparticles demonstrates an eco-friendly method for producing nanomaterials. Conventional techniques for synthesizing titanium dioxide nanoparticles (TiO₂ NPs) frequently rely on high-temperature sol–gel processes or flame pyrolysis, which typically produce particles larger than 100 nm, as well as hydrothermal methods [34].

Zinc oxide nanoparticles (ZnO NPs) are typically produced using methods such as high-pH precipitation or the thermal decomposition of zinc salts. In contrast, our biosynthetic technique was carried out in an aqueous solution at temperatures between 30 and 37 °C, utilizing microbial metabolism to facilitate the formation of particles. The titanium dioxide nanoparticles (TiO₂ NPs) we synthesized, measuring approximately 70 nm and consisting of a mixed phase, are similar in size to those documented by previous studies. For biosynthesized TiO₂ using Bacillus subtilis, where they reported particle sizes ranging from 80 to 120 nm as observed through scanning electron microscopy (SEM). Additionally, those researchers also noted the presence of mixed anatase and rutile phases in their synthesis [35].

Similarly, Our XRD results indicate that microbial synthesis can lead to the production of a polycrystalline material. Notably, B. subtilis appears to facilitate the formation of rutile at comparatively low temperatures. In contrast, traditional chemical methods typically yield only anatase unless the materials are calcined at temperatures exceedingly approximately 600 °C. This difference may be attributed to the presence of specific biomolecules or fluoride, particularly in studies where TiF₆²⁻ was used as a precursor, which may facilitate the templating of the rutile structure. Furthermore, our experiments using TiOSO₄ combined with mild annealing at 200 °C resulted in the inclusion of rutile, indicating that rutile nuclei may have formed during the initial biosynthesis phase, potentially catalyzed by organic residues [36].

The zinc oxide nanoparticles (ZnO NPs) derived from Bacillus are notably diminutive, measuring approximately 18 nanometers in size, and exhibit a well-defined wurtzite structure. Sabir et al. documented the biosynthesis of ZnO using Bacillus sp., reporting particle dimensions ranging from 16 to 20 nanometers as observed through transmission electron microscopy (TEM), which correlates strongly with our measurements. This consistent nanoscale dimension is likely attributed to the capping influence of bacterial proteins that inherently restrict crystal growth. Furthermore, the specific conditions of the bacterial environment, characterized by fluctuations in pH and the gradual conversion of precursors, promote the emergence of numerous nucleation sites, consequently leading to the formation of smaller particles. The high purity of our synthesized ZnO is evident, as there are no impurity peaks in the X-ray diffraction (XRD) analysis, and the energy dispersive spectrometry (EDS) results confirm the presence of only zinc and oxygen elements, aligning with other environmentally friendly synthesis methods that typically yield relatively pure metal oxides. An important aspect to consider is that these biogenic nanoparticles are enveloped in a biocompatible coating composed of proteins and polysaccharides [37].

In specific contexts, this characteristic can prove beneficial, particularly in fields such as nanomedicine, where biocompatibility is crucial for effective treatment. However, in the realm of photocatalysis, the scenario becomes more complex. On one side, the remaining organic compounds on titanium dioxide (TiO₂) may serve as sensitizers or dopants, capable of absorbing visible light and facilitating the injection of electrons into the TiO₂, much like the dye-sensitization process. Conversely, these same organic materials can interfere with the photocatalytic process by capturing the radicals generated by light exposure, thereby diminishing the effectiveness of antibacterial actions [38].

Under our experimental conditions, no significant phototoxicity was detected in E. coli upon UV exposure, which we attribute to the presence of residual organic coatings on the nanoparticles that scavenged reactive oxygen species (ROS). However, UV irradiation is well documented to cause DNA damage and genotoxic stress in bacteria under standard conditions [39].

In this scenario, the pronounced antimicrobial action observed in the presence of light indicates that the leftover organic materials were likely insufficient to neutralize all reactive oxygen species (ROS). Alternatively, the gentle heat treatment effectively eliminated a sufficient amount of these organics to achieve an equilibrium between stability and antimicrobial activity [40].

The FTIR confirms organics are present, so some ROS might indeed be consumed. Perhaps the superior performance of the combined TiO₂/ZnO coating is partly because ZnO (which is also capped with organics) generates H₂O₂ under light that can diffuse out and is not completely scavenged. It is also possible that during the 24 h incubation on agar, some biodegradation of the capping occurred, freeing the NP surfaces [41].

4.2. Visible-Light Photocatalysis

The attainment of antimicrobial efficacy under visible light represents a significant advancement in this study. Traditional pure TiO₂ (anatase) exhibits minimal absorption beyond 400 nm, thereby rendering it typically inactive under fluorescent lighting conditions. However, the TiO₂ developed in this research demonstrates a band gap of approximately 2.9 eV, enabling absorption that extends to around 430 nm. Several factors can elucidate this phenomenon: (i) The presence of both anatase and rutile phases—rutile, characterized by a lower band gap of roughly 3.0 eV, effectively shifts some absorption into the 410 nm and above range. Additionally, the junction formed between anatase and rutile enhances charge transfer capabilities in the visible light spectrum. (ii) The introduction of carbon and nitrogen doping is another contributing factor; organic residues originating from Bacillus may introduce these elements into the TiO₂ lattice. For instance, during the drying process at 200 °C, the decomposition of organic matter could lead to surface doping. Prior research indicates that carbon-doped TiO₂ can absorb visible light, with band gaps ranging from approximately 2.7 to 2.9 eV. Evidence supporting slight doping includes the observed absorption tail and the light-grey coloration of the TiO₂ powder. (iii) The formation of defect states, such as oxygen vacancies or Ti³⁺ centers during the biosynthesis process, may create mid-gap energy levels that facilitate the absorption of visible wavelengths [42].

Zinc oxide (ZnO), primarily recognized for its role as a UV absorber, exhibits the capability to produce visible luminescence and reactive oxygen species (ROS) when defects, such as oxygen vacancies, are present —a characteristic frequently observed in nanocrystalline ZnO. The interaction of ZnO with blue light excitation may thus yield considerable photochemical activity. Additionally, fluorescent lamps generally emit a slight UV component, approximately less than 5% within the 350–400 nm range, which can sufficiently excite ZnO, suggesting that it is not entirely inactive in the visible spectrum. Under ambient light conditions, ZnO likely produces peroxides to some extent.

Moreover, it is crucial to acknowledge that ZnO exhibits partial solubility in aqueous environments, particularly when carbon dioxide is present, leading to the possible release of Zn²⁺ ions and zincate species under alkaline conditions. This ion release may contribute to the modest antibacterial activity observed in dark conditions. While this mechanism is consistent with prior reports of Zn²-mediated toxicity, the present study did not include direct quantification of Zn²⁺ leaching. Future work will incorporate ICP-OES or ICP-MS measurements to provide quantitative confirmation of Zn²⁺ release from the coatings and its correlation with antibacterial activity [43].

The interaction between TiO₂ and ZnO when exposed to visible light is particularly noteworthy. Comparable observations have been documented regarding TiO₂–ZnO composites: A research investigation revealed that a TiO₂/ZnO nano-hybrid exhibited a more pronounced antibacterial effect compared to each component acting independently. This finding indicates that the synergistic interaction between the ion release from ZnO and the photocatalytic properties of TiO₂ produces a highly effective antibacterial agent [44].

The multilayer structure can be described as a nano-hybrid coating. When exposed to light, electron-hole pairs are generated in both semiconductors present in the coating. The conduction band (CB) edge of ZnO is approximately −0.5 V (versus Normal Hydrogen Electrode), while the CB edge of TiO₂ in its anatase phase is around −0.3 V. This difference allows excited electrons from ZnO to transfer to TiO₂, effectively separating positive and negative charges at the interface. This separation facilitates the formation of more reactive oxygen species (ROS): electrons from TiO₂ reduce oxygen to superoxide, while holes in ZnO oxidize water to hydroxyl radicals (•OH), among other reactions. Furthermore, any hydrogen peroxide (H₂O₂) produced by these processes can diffuse freely and interact with bacteria. The multilayer structure may also enhance light reflection and scattering, potentially increasing the opportunities for photon absorption. While such effects could be consistent with photonic crystal or stop-band behavior, no reflectance or transmittance spectra were obtained in this study to confirm this phenomenon directly. Therefore, we cautiously interpret the observed iridescence and refractive index contrast as suggestive of enhanced light trapping, which warrants further verification in future optical studies. Although a minimal contribution of UVA radiation (350–420 nm) cannot be entirely excluded, the fluorescent lamp used in this study was specified by the manufacturer to emit negligible irradiance (<5%) in this range. Therefore, the antibacterial activity observed can be attributed primarily to visible-light photocatalysis [42,45].

4.3. Optical Evidence of Light Trapping

The observed red-shift in TiO₂ absorption and defect-tail absorption in ZnO suggest that biogenic nanoparticles inherently broaden the spectral response into the visible region. The multilayer architecture further amplifies this effect by combining the complementary absorption profiles and increasing scattering/interaction length within the stack. Although direct reflectance spectra of the coated slides were not measured, the enhanced H₂O₂ production quantified by FOX provides functional evidence of improved light harvesting. The multilayer generated two-fold higher ROS than ZnO and nearly six-fold higher than TiO₂ alone, which mirrors its superior antibacterial performance. These converging indicators—band-gap shift, ROS quantification, and inhibition zone size—support the claim that the multilayer structure enhances photon utilization under visible-light illumination [27,42].

4.4. Mechanistic Confirmation by Radical Scavenging

The radical scavenging experiments provide strong evidence that the antibacterial activity of TiO₂/ZnO coatings under visible light is mediated primarily through ROS generation. The marked reduction in inhibition zones upon the addition of mannitol, catalase, and p-benzoquinone indicates the involvement of hydroxyl radicals (·OH), hydrogen peroxide (H₂O₂), and superoxide radicals (O₂·⁻), respectively. These findings are consistent with the photocatalytic mechanism proposed for TiO₂/ZnO heterojunctions, in which photoexcited electrons and holes facilitate the generation of diverse ROS. The greater reduction observed in the multilayer coating compared to individual TiO₂ or ZnO layers further confirms that the enhanced antibacterial effect originates from synergistic ROS production and improved charge separation at the heterointerface. Together, these results validate the claim that the antibacterial action of the coatings is predominantly “visible-light-driven” and mechanistically dependent on ROS-mediated disinfection [4].

4.5. Role of ROS Generation

Direct measurement of H₂O₂ under visible light provided functional evidence that the antibacterial mechanism of the TiO₂/ZnO multilayer is photocatalytic and ROS-mediated. The significantly higher H₂O₂ levels detected for the multilayer compared to single-component coatings support the synergistic role of TiO₂–ZnO heterojunctions in enhancing charge separation and reactive oxygen production. The negligible H₂O₂ detected in dark conditions further confirms that antibacterial activity is not due to passive ion release but is light-driven. These findings also clarify the contribution of the bio-organic capping observed in FTIR. Although residual biomolecules could theoretically quench ROS, the strong H₂O₂ output under illumination indicates that their effect is either minimal or compensated by sensitization/doping effects that narrow the band gap and extend visible-light absorption. Overall, the FOX assay results reinforce the proposed mechanism in which visible-light irradiation activates the TiO₂/ZnO multilayer to generate diffusible ROS such as H₂O₂, which correlates with bacterial inactivation in both disk diffusion and direct contact assays [12].

4.6. Validation of Contact-Killing Ability

While the disk diffusion assay primarily reflects the activity of soluble or diffusible components, the direct contact-killing assay provides a more rigorous assessment of the antibacterial properties of fixed coatings. Our results demonstrate that the TiO₂/ZnO multilayer nanocoating achieved significant log reductions in viable bacteria upon direct surface exposure under visible light, confirming its capacity for contact-mediated killing. The higher activity observed against S. aureus compared to E. coli is consistent with differences in cell wall structure and permeability.

These findings highlight that the antibacterial efficacy of the multilayer coating arises not only from diffusible ROS and Zn²⁺ species but also from direct interactions at the coating–bacteria interface. This dual mode of action—contact killing and photocatalytic ROS generation—further strengthens the potential of TiO₂/ZnO multilayer coatings for practical antimicrobial surface applications in healthcare environments [6,28].

4.7. Antibacterial Activity Compared to Literature

The ~15 mm inhibition zone for S. aureus and ~12 mm for E. coli achieved by the TiO₂/ZnO multilayer under visible light falls within the general range reported for chemically synthesized ZnO (10–15 mm for both S. aureus and E. coli) [46]. It should be noted, however, that the nanoparticle concentration used in the cited study was not specified; therefore, this comparison is qualitative rather than quantitative and is provided only to indicate consistency with the broader literature. TiO₂ alone showed negligible activity unless UV is used, consistent with the literature [47,48,49].

The ~3 mm zone observed for TiO₂ under visible light may reflect a slight reduction in the band gap. ZnO’s 9–10 mm zone aligns with its known UVA-driven activity and Zn²⁺ toxicity. Notably, the multilayer effect exceeded additive expectations (~13 mm), suggesting synergistic interaction.

This synergy likely stems from the enhanced production of hydrogen peroxide (H₂O₂) under light by both TiO₂ and ZnO. Acting as photocatalytic “chemical factories,” these coatings release H₂O₂ into surrounding media, expanding their antimicrobial reach beyond direct contact [50,51].

It is worth mentioning that Bacillus subtilis, a microorganism commonly employed in the biosynthesis of nanoparticles (NPs), has also been used as a test organism in studies evaluating the antimicrobial activity of titanium dioxide NPs against both B. subtilis and Escherichia coli. Their experiments revealed a zone of inhibition of approximately 12 mm for B. subtilis and around 16 mm for E. coli, which contrasts with our observations regarding the Gram classification of bacteria. Notably, they administered a higher concentration of NPs to the E. coli strains, as indicated in their paper, which suggested that E. coli required a greater NP dosage to manifest an effect. This factor could account for the larger inhibition zone observed in their findings. This emphasizes the notion that adequate NP loading can effectively counteract the resistance exhibited by Gram-negative bacteria. In our research, the multilayer structure likely facilitated a higher effective dosage of both reactive oxygen species (ROS) and zinc ions (Zn²⁺) delivered to E. coli, resulting in a respectable zone of inhibition, albeit slightly smaller than that observed for Staphylococcus aureus [52].

4.8. Stability and Durability Considerations

While the primary aim of this work was to demonstrate the visible-light antibacterial performance of biogenic TiO₂/ZnO multilayers, stability is an equally critical parameter for clinical translation. Our preliminary washing and wiping tests indicated that the coatings remained adherent to glass substrates without visible delamination, suggesting acceptable short-term durability. This outcome is consistent with the layer-by-layer architecture and mild thermal treatment, which promote particle interlinking and adhesion. Nevertheless, comprehensive mechanical testing (tape adhesion, abrasion resistance, and repeated cleaning/disinfection cycles) was not conducted within this study. We acknowledge this as a limitation and emphasize that evaluating long-term durability under realistic hospital cleaning protocols will be a primary focus of future investigations [27,28].

4.9. Implications for Hospital Surfaces

Demonstrating antimicrobial activity under visible light offers practical value for hospital settings. Standard lighting (LED or fluorescent) can continuously activate these coatings, reducing surface contamination without the risks and costs associated with UV systems. The biosynthesis method using Bacillus subtilis is low-cost, scalable, and environmentally friendly [53], making large-scale production feasible for coatings, paints, or sprays. Although the current study employed a multilayer model on glass, future applications could involve embedding nanoparticles in polymer matrices or utilizing sol–gel films to enhance durability and adhesion. This strategy could be extended to common surfaces, such as steel or plastic, using dip or spray coating with post-curing.

4.10. Safety and Efficacy

The substances employed (TiO₂ and ZnO) are widely regarded as safe for topical use and environmental exposure. ZnO is incorporated into wound dressings and sunscreens, while TiO₂ is found in a variety of consumer products. Nonetheless, the nanoparticle form raises valid concerns related to potential inhalation or ingestion should particles become dislodged. In a clinical context, secure adhesion of the coating is therefore essential to minimize this risk. An additional benefit of our biogenic nanoparticles is their intrinsic capping with biomolecules derived from Bacillus subtilis, which may mitigate acute cytotoxicity. Supporting this, Mansoor et al. reported that TiO₂ nanoparticles biosynthesized by B. subtilis exhibited no detectable cytotoxicity when incorporated into a dental composite at a concentration of 5%, suggesting that such materials can be used safely in applications involving human contact. Nevertheless, we emphasize that nanoparticle safety is dose- and context-dependent, and that comprehensive biocompatibility testing—including inhalation and chronic exposure studies—remains necessary prior to clinical translation. Importantly, the byproducts of photocatalysis are primarily water and CO₂ (arising from mineralization of organic matter and microbial cells), which at the levels generated pose no recognized health risk [54].

4.11. Role of Zn²⁺ Release and Bacterial Tolerance

In addition to photocatalytically generated ROS, partial dissolution of ZnO can release Zn²⁺ ions that contribute to antibacterial activity. The higher sensitivity of S. aureus compared to E. coli is consistent with their cell envelope structures: the thick but permeable peptidoglycan of S. aureus facilitates Zn²⁺ uptake, whereas the Gram-negative outer membrane and efflux systems of E. coli restrict ion penetration. The apparent survival of Bacillus subtilis during biosynthesis can be explained by the extracellular synthesis context, where nutrient-rich media buffer and precipitate Zn²⁺ as Zn (OH)₂, and by intrinsic metal tolerance mechanisms in B. subtilis. These include the Zur regulon, which tightly regulates zinc uptake genes [55], and protein/ligand coordination dynamics that buffer intracellular Zn²⁺ and maintain sub-toxic free ion levels [56,57]. Thus, the contrast between synthesis conditions and antibacterial assays underscores the dual contribution of localized Zn²⁺ release and ROS generation in determining bactericidal efficacy.

4.12. Comparison with Other Approaches

There are alternative methods for creating antimicrobial surfaces that are activated by visible light, including the use of dye-sensitized TiO₂ (for instance, TiO₂ that is coated with a dye that absorbs visible light) and employing graphitic carbon nitride (g-C₃N₄) as a visible-light photocatalyst. The method we propose, which utilizes inherently doped TiO₂ and ZnO, offers a straightforward solution. Additionally, its self-sterilizing properties ensure that the coating remains intact and is not depleted over time, unlike antimicrobial polymers that may leach. Dye-sensitized surfaces are subject to degradation as the dye is consumed, whereas our inorganic nanoparticles serve as regenerative catalysts, continuously functioning as long as light and oxygen are available. Future research comparing our bio-nanocoating to commercial photocatalytic coatings, such as Cu-doped TiO₂ or N-doped TiO₂, would provide valuable insights into differences in effectiveness.

In conclusion, our method of creating biogenic TiO₂/ZnO nanocoatings has demonstrated its practicality and effectiveness. This approach aligns with the increasing trend of utilizing nanomaterials synthesized through microbial processes for innovative applications. The combination of these materials overcomes the constraint of pure TiO₂, which requires UV light, by utilizing the capabilities of hospital lighting to promote photocatalytic disinfection when the materials are appropriately designed. This study establishes a basis for the experimental implementation of such coatings. This suggests that environmentally friendly methods for fabricating nanoparticles can be combined with intelligent coating strategies to provide viable solutions for combating hospital-acquired infections.

5. Conclusions

This study demonstrates that biogenically synthesized TiO₂ and ZnO nanoparticles, produced using Bacillus subtilis, can be successfully integrated into multilayer photonic coatings with potent visible-light-driven antibacterial properties. The coatings exhibited strong inhibition against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria, with inhibition zones reaching ~15 mm, clearly outperforming single-component coatings. The enhanced activity arises from synergistic mechanisms, including band-gap narrowing of TiO₂ (2.9 eV), partial ZnO activation under blue light, and cooperative effects of ROS generation and Zn²⁺ release.

Notably, the coatings were highly effective under typical indoor lighting while remaining largely inactive in darkness, highlighting their unique “on-demand” antimicrobial functionality. This visible-light responsiveness provides a safe, sustainable, and practical alternative to UV-based disinfection strategies.

The findings establish a proof-of-concept for eco-friendly, scalable nanocoatings capable of reducing hospital-acquired infections on frequently touched surfaces and surgical instruments. Future work should focus on durability testing under hospital cleaning protocols, safety assessments, and exploring antiviral applications, potentially extending their use to the inactivation of enveloped viruses such as SARS-CoV-2.