Interfacial Energy Analysis of Lubricant Stability and Anti-Biofouling Performance of Slippery Liquid-Infused ZnO Nanostructured Surfaces

Abstract

This paper investigated the lubricant stability and anti-biofouling performance on slippery liquid-infused nanostructured surfaces (SLIPSs) using thermodynamic and interfacial energy analyses. ZnO nanorods were synthesized on indium tin oxide substrates with and without a ZnO seed layer by electrochemical deposition and subsequently infused with PDMS-based silicone oil. The seed-layer-assisted growth produced densely packed and vertically aligned ZnO nanorods. Interfacial energy analysis showed that the resulting SLIPSs satisfied the criterion for resistance to water-induced lubricant displacement (ΔE₂ = 64.14 mJ·m⁻²), indicating effective lubricant retention, whereas non-seeded surfaces exhibited reduced lubricant stability. Consistent with this prediction, the seed-layer-assisted SLIPSs retained droplet mobility following spin testing at 2500 rpm, although the reduced sliding velocity indicated a decline in slippery performance. Anti-biofouling evaluation using Escherichia coli (XL1-Blue) revealed that the SLIPSs effectively suppressed bacterial attachment, reducing surface coverage to below 0.3% after 24 h of incubation. In comparison, the pristine ITO and ZnO nanorods exhibited values of 50.8% and 54.6%, respectively. Subsequent surface free energy analysis demonstrated that lubricant infusion reduced the work of adhesion to 77.92 mJ·m⁻². These findings provide insight into the interfacial interactions governing lubricant retention and bacterial attachment on SLIPSs.

1. Introduction

Bacterial adhesion serves as the pivotal initial stage of colonization, which is driven by complex physicochemical interactions that precipitate subsequent biofilm formation [1]. This phenomenon, known as biofouling, involves the undesirable accumulation of microorganisms, including bacteria, fungi, and marine biofouling organisms. It causes persistent contamination and degrades surface performance in both biomedical and industrial fields [2,3,4]. Conventional biocidal coatings have long been employed to prevent microbial colonization. However, their use poses environmental risks due to the release of toxic agents that harm non-target organisms [5,6].

To address these challenges without relying on harmful chemicals, bio-inspired surface strategies have gained traction, drawing direct inspiration from the slippery mechanics of the Nepenthes pitcher plant. The peristome’s microstructures enable the capillary entrapment of a lubricating liquid, forming a stable continuous film that prevents direct solid–liquid contact. This mechanism serves as the fundamental principle underlying the development of slippery liquid-infused porous surfaces [7,8]. Aizenberg et al. pioneered the SLIPS concept by infusing perfluorinated liquids into nanofibrous Teflon membranes, demonstrating exceptional liquid repellency and robust high-pressure stability [9]. Recent studies have shown that SLIPSs reduce biofilm surface coverage by more than 90% compared with untreated substrates, highlighting their significant potential for anti-biofouling applications [10,11].

Despite the exceptional performance of SLIPSs, lubricant depletion or liquid displacement can deteriorate their liquid-repellent properties under practical conditions. This degradation exposes the underlying solid substrate, thereby increasing susceptibility to bacterial attachment [8]. Previous studies have primarily evaluated SLIPS performance using macroscopic parameters such as contact angle hysteresis, sliding angle, and bacterial surface coverage [12,13]. Although these parameters provide useful insights into surface wettability and anti-biofouling performance, the combined consideration of lubricant stability and bacterial adhesion remains insufficiently elucidated. The interactions between the lubricant and the solid substrate play a critical role in stabilizing the lubricating layer. At the same time, bacterial attachment is influenced not only by surface wettability but also by surface free-energy components, particularly polar and acid–base interactions [14]. Therefore, considering lubricant stability together with component-specific surface free energy and bacterial adhesion can provide additional insight into the thermodynamic interactions at the bacteria–surface interface. In this paper, these aspects are integrated to provide insights into lubricant stability and anti-biofouling performance.

Nanostructured materials with tunable features enable the systematic exploration of how surface morphology affects lubricant stability. Recent studies have also demonstrated the important role of surface microstructure in regulating liquid-spreading behavior [15]. ZnO is a promising scaffold for SLIPSs due to its high surface area and morphological tunability [16]. Crucially, pre-coated seed layers enable the precise control of surface topography, creating an ideal platform for studying capillary confinement. By accommodating infused lubricants within its geometric structure, ZnO provides an excellent model system for investigating lubricant retention in liquid-infused surfaces. A previous ZnO-based SLIPS study demonstrated the influence of ZnO nanostructure on lubricant retention and slippery-surface performance [17].

In this paper, slippery liquid-infused ZnO nanostructured surfaces were fabricated to systematically examine the effect of controlled surface topography on lubricant stability and anti-biofouling performance. Interfacial energy analyses, based on surface free energy components, were employed to evaluate the thermodynamic criteria governing lubricant retention and bacterial adhesion. These findings provide critical mechanistic insight into the coupling between surface energetics and SLIPS performance.

2. Materials and Methods

2.1. Preparation of the ZnO Seed Layer

The fabrication process of the ZnO-based slippery liquid-infused nanostructured surfaces (SLIPSs) is schematically illustrated in Figure 1. Indium tin oxide (ITO)-coated glass substrates (<10 Ω/sq, WELLJOIN, Taipei, Taiwan) were cut into 2 × 2 cm² pieces and sequentially sonicated in acetone (CH₃COCH₃, ≥99.8%, Merck, Darmstadt, Germany), isopropanol (C₃H₈O, 99.7%, Carlo Erba Reagents, Cornaredo, Italy), and deionized water (resistivity of 2 MΩ·cm) for 20 min per solvent to remove surface contaminants; then, they were dried under a N₂ flow. The ZnO seed layer precursors were prepared by dissolving 10 mM zinc acetate dihydrate (Zn(CH₃CO₂)₂·2H₂O, ≥99%, J.T. Baker, Phillipsburg, NJ, USA) and 10 mM monoethanolamine (NH₂CH₂CH₂OH, 99%, Carlo Erba Reagents, Cornaredo, Italy) in isopropyl alcohol, which was followed by stirring for 3 h to achieve homogeneity. For the sol–gel deposition, the mixture was spin-coated onto the cleaned substrates at 2500 rpm for 60 s. This cycle was repeated four times to ensure film uniformity. Finally, the samples were annealed at 300 °C for 1 h to eliminate organic residues and to enhance the adhesion and densification of the ZnO seed layer.

Figure 1. Schematic illustration of the ZnO nanostructure-based SLIPS fabrication.

2.2. Growth of ZnO Nanostructures on a Template

To generate the surface roughness required for SLIPS fabrication, ZnO nanostructures were grown on the pre-deposited seed layers via electrochemical deposition. The electrochemical cell was configured using the ITO-coated glass substrate as the cathode and a zinc foil as the anode. The aqueous electrolyte was prepared by dissolving 10 mM zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O, 98%, Ajax Finechem, Seven Hills, Australia) and 10 mM hexamethylenetetramine (C₆H₁₂N₄, 99.5%, HiMedia Laboratories, Thane, India) in deionized water and then stirring for 5 min to ensure homogeneity. The electrolyte was preheated at 80 °C for 15 min and maintained at this temperature during the growth process. Electrodeposition was carried out for 40 min at a constant electrical potential of 0.5 V with an electrode spacing of 2.0 cm.

2.3. Fabrication of Slippery Liquid-Infused Nanostructured Surfaces (SLIPSs)

Following ZnO nanostructure growth, the samples were immersed in a PDMS-based silicone oil (polydimethylsiloxane, [–Si(CH₃)₂–O–]_n, 50 cSt, Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 12 h. To facilitate oil penetration and minimize trapped air within the nanostructures, the immersion vessel was placed under vacuum using a rotary pump. Subsequently, the samples were positioned vertically for 1 h to allow excess lubricant to drain under gravity, thereby stabilizing the liquid-infused nanostructured surfaces (SLIPSs) before characterization.

2.4. Characterization

2.4.1. Morphological, Topographical, and Structural Characterization

The surface morphology and topography of the ZnO nanostructured surfaces were investigated via field-emission scanning electron microscopy (FE-SEM; Nova NanoSEM 450, FEI Company, Hillsboro, OR, USA) and atomic force microscopy (AFM; Park NX-10, Park Systems, Suwon, Republic of Korea), respectively. Crystallographic structures were analyzed using X-ray diffraction (XRD; Smart Lab SE, Rigaku Corporation, Tokyo, Japan) with a 2θ scanning range of 20–80°.

2.4.2. Surface Wettability and Slippery Surface Performance

The wettability of the ZnO nanostructured surfaces and SLIPSs was evaluated using an optical contact angle goniometer (SL150E, KINO Scientific Instrument Inc., Boston, MA, USA) with CAST^®3.0 software for drop shape analysis. Static contact angles were recorded using 4 µL deionized water droplets across five different locations per sample to verify uniformity. Surface free energy components (${\mathsf{\gamma }}^{\mathrm{LW}}$, ${\mathsf{\gamma }}^{+}$ and ${\mathsf{\gamma }}^{-}$) were determined via the van Oss–Chaudhury–Good (vOCG) approach using three probe liquids (surface tension parameters detailed in Table 1). Droplet mobility was quantified by measuring water sliding velocities at a 10° tilt angle. Finally, the durability of the lubricant-infused surfaces was comparatively assessed by spinning samples at various rotational speeds with performance retention evaluated by comparing the sliding velocities before and after the spin test.

Table 1. Lifshitz–van der Waals, acid–base, and total surface tension components of the test liquids (at 20 °C) used in this paper [18].

Liquid	Surface Tension (mJ·m−2)
	${\mathsf{\gamma }}^{\mathbf{TOT}}$	${\mathsf{\gamma }}^{\mathbf{LW}}$	${\mathsf{\gamma }}^{\mathbf{AB}}$	${\mathsf{\gamma }}^{+}$	${\mathsf{\gamma }}^{-}$
Water	72.8	21.8	51.0	25.5	25.5
Ethylene glycol	48.0	29.0	19.0	1.92	47.0
Diiodomethane	50.8	50.8	0	0	0

2.4.3. Optical Analysis and Anti-Biofouling Evaluation

The optical properties of ZnO nanostructures and silicone oil were investigated using UV–Vis absorption and photoluminescence (PL) spectroscopy to evaluate their intrinsic emission and ensure compatibility with fluorescence imaging. Anti-biofouling performance was assessed using Escherichia coli (XL1-Blue) suspensions in Luria–Bertani (LB) broth, which were provided by a collaborating microbiology laboratory. For adhesion assays, bare ITO, ZnO nanostructures, and SLIPS samples were placed at a slight incline, and the bacterial suspension was drop-cast onto each surface. Following incubation at 37 °C for 1, 3, and 24 h, non-adherent bacteria were gently rinsed away with deionized water, and the samples were air-dried under ambient conditions. Bacterial colonization was subsequently analyzed via confocal laser scanning microscopy (CLSM) with surface coverage quantified using fluorescence image analysis.

3. Results and Discussion

3.1. Influence of ZnO Seed Layer on the Morphology and Crystal Structure of ZnO Nanorods

Figure 2a shows the surface morphology of the ZnO seed layer deposited on the ITO-coated substrate. Owing to the thinness of the seed layer and the underlying ITO substrate, resolving individual features remains challenging. However, the FE-SEM image confirms homogeneous substrate coverage via the spin-coating process. To better resolve the surface topography, atomic force microscopy (AFM) was employed (Figure 2b), revealing a root-mean-square roughness (R_q) of 6.4 nm. This nanoscale roughness provides a uniform template that facilitates subsequent ZnO nanorod nucleation.

Figure 2. (a) FE-SEM and (b) AFM images of ZnO seed layer along with (c) top-view and (d) cross-sectional FE-SEM images of the ZnO nanorods grown at 80 °C for 40 min.

Figure 2c,d revealed that ZnO nanorods form a dense array with a preferential vertical orientation on the substrate. This morphology indicates effective nucleation on the underlying ZnO seed layer, yielding well-defined nanorods with an average diameter of 58.92 ± 7.54 nm and a length of 897.99 ± 9.58 nm.

Figure 3 presents the diameter distributions of ZnO nanorods grown with and without a pre-coated seed layer, which were derived from a statistical analysis of top-view FE-SEM images. Without the seed layer, the nanorods exhibit broader, less uniform diameters with an average of 273.62 ± 40.22 nm. Quantitative analysis reveals a nanorod density of 171.20 ± 15.53 rods·µm⁻² on the seed layer, whereas a significantly lower density of 3.14 ± 0.30 rods·µm⁻² occurs on the bare substrate. This stark contrast stems from distinct nucleation behaviors. The ZnO seed layer facilitates more uniform nucleation, promoting vertical alignment while suppressing lateral growth [19]. Conversely, a scarcity of nucleation sites on the bare substrate forces competitive lateral growth, leading to larger diameters, random orientations, and incomplete surface coverage.

Figure 3. Diameter distributions of ZnO nanorods grown on (a) seed-coated and (b) bare ITO/glass substrates.

Figure 4 presents the XRD patterns for the seed layer and the grown ZnO nanorods. For the seed layer, the diffractogram is dominated by peaks at 2θ ≈ 21.3°, 30.3°, 35.2°, 50.6°, and 60.2° (Figure 4b), corresponding to the (211), (222), (400), (440), and (622) planes of cubic In₂O₃ (JCPDS card no. 65-3170) from the ITO substrate. This dominance stems from the thin ZnO seed layer, which allows X-rays to penetrate into the underlying substrate [20].

Figure 4. XRD patterns of (a) all samples, (b) the ZnO seed layer (black line), (c) ZnO NRs on seed-coated ITO/glass (red line), and (d) ZnO NRs on bare ITO/glass (blue line).

For the unseeded nanorods, Figure 4c exhibits several diffraction peaks at 2θ ≈ 31.8°, 34.4°, and 36.3°, indexing to the (100), (002), and (101) planes of wurtzite ZnO (JCPDS no. 36-1451), respectively. In contrast, ZnO nanorods synthesized on the seeded substrate exhibit a highly dominant (002) reflection at 34.4° with other peaks significantly suppressed (Figure 4d). This high-intensity (002) peak indicates a strong preferential c-axis orientation perpendicular to the substrate, highlighting the critical role of the seed layer in directing the oriented nanorod growth [21].

3.2. Wettability and Droplet Mobility of Lubricant-Infused ZnO Nanorod Surfaces

Figure 5 presents the water contact angles of samples S1–S3 measured before and after lubricant infusion. Before infusion, bare ITO (S1) exhibited a low contact angle of approximately 35.1°, which was attributable to its intrinsically high surface free energy and inherent polarity. The unseeded and seeded ZnO nanorod surfaces (S2 and S3) displayed markedly different wetting behaviors. S2 exhibited a hydrophobic contact angle of 137.4°, whereas S3 presented a hydrophilic value of 28.5°. This divergence arises from variations in the nanorod alignment and surface morphology [22,23]. The seed layer promotes the vertical alignment of ZnO nanorods, which increases the exposure of high-surface-energy polar (0001) facets to the water droplet. Conversely, the absence of a seed layer leads to random orientations, enhancing the relative exposure of lower-energy non-polar $\{10\overline{1}0\}$ facets.

Figure 5. Water contact angles of S1 (bare ITO/glass), S2 (ZnO nanorods without a seed layer), and S3 (ZnO nanorods with a seed layer) before and after silicone oil infusion with error bars representing 5 measurements across 5 locations.

Upon lubricant infusion, the contact angles of all samples converged to approximately 100°. This shift is attributed to the non-polar methyl (–CH₃) groups and low surface tension of the silicone oil. These results demonstrate that the wettability of the liquid-infused surfaces is predominantly dictated by the lubricant rather than the underlying substrate. Because the water droplet rests on the oil layer, it forms a liquid–liquid interface rather than a direct solid–liquid contact with the ZnO surface [7]. The structural role of the ZnO nanorods in retaining this lubricant layer is evaluated in the subsequent section.

To evaluate dynamic liquid repellency, the mobility of water droplets on infused samples S1–S3 was examined. Figure 6 presents time-lapse images of droplets on the surfaces tilted at 10°. Droplets on S1 and S2 remained pinned under this condition, indicating that lubricant retention depends heavily on surface architecture. The smooth ITO surface (S1) lacks the capillary features required to act as a lubricant reservoir, while the disordered nanorod alignment of S2 provides insufficient oil confinement. In contrast, S3 demonstrated steady droplet motion at the same tilt angle, achieving an average sliding velocity of 0.74 ± 0.05 mm·s⁻¹. The vertically aligned nanorod array of S3 promotes uniform capillary confinement of the infused silicone oil, enabling a stable lubricating layer. The thermodynamic stability governing this behavior is evaluated below based on specific energy criteria [9,24,25]:

$${∆E}_1=R({\mathsf{\gamma }}_{\mathrm{B}}\mathit{cos}{\mathsf{\theta }}_{\mathrm{B}}-{\mathsf{\gamma }}_{\mathrm{A}}\mathit{cos}{\mathsf{\theta }}_{\mathrm{A}})+{\mathsf{\gamma }}_{\mathrm{A}}-{\mathsf{\gamma }}_{\mathrm{B}}>0$$

(1)

$${∆E}_2=R({\mathsf{\gamma }}_{\mathrm{B}}\mathit{cos}{\mathsf{\theta }}_{\mathrm{B}}-{\mathsf{\gamma }}_{\mathrm{A}}\mathit{cos}{\mathsf{\theta }}_{\mathrm{A}})-{\mathsf{\gamma }}_{\mathrm{AB}}>0$$

(2)

where ${\mathsf{\gamma }}_{\mathrm{A}}$, ${\mathsf{\gamma }}_{\mathrm{B}}$ and ${\mathsf{\gamma }}_{\mathrm{AB}}$ are the surface tensions of deionized water (${\mathsf{\gamma }}_{\mathrm{A}} = 72.8$ mJ·m⁻²), silicone oil (${\mathsf{\gamma }}_{\mathrm{B}} = 21.0$ mJ·m⁻²), and the interfacial tension between water and oil (${\mathsf{\gamma }}_{\mathrm{AB}} = 36.0$ mJ·m⁻²), respectively [26]. ${\mathsf{\theta }}_{\mathrm{A}}$ and ${\mathsf{\theta }}_{\mathrm{B}}$ are the contact angles of water and oil on a flat solid surface, respectively. R is the roughness factor (ratio of the actual to the projected surface area).

Figure 6. Time-lapse images of water droplets sliding on silicone oil-infused samples (S1–S3).

The calculated values in Table 2 provide a thermodynamic basis for the liquid-repellent behavior of the fabricated surfaces. All samples satisfy the first stability criterion (ΔE₁ > 0), confirming that lubricant infusion is energetically favorable in air. However, their resistance to water-induced lubricant displacement varies substantially. The negative ΔE₂ values for S1 and S2 indicate thermodynamically unstable lubricant layers, which are consistent with the droplet pinning observed in Figure 6. In contrast, S3 exhibits a positive ΔE₂ value of 64.14 mJ·m⁻², demonstrating robust thermodynamic resistance to water-induced lubricant displacement.

Table 2. Surface parameters and calculated thermodynamic stability criteria (ΔE₁ and ΔE₂) for samples S1–S3.

Sample	Sample Types	R	${\mathsf{\theta }}_{\mathbf{A}}$ (deg.)	${\mathsf{\theta }}_{\mathbf{B}}$ (deg.)	$∆{\mathit{E}}_1$ (mJ·m−2)	$∆{\mathit{E}}_2$ (mJ·m−2)
S1	ITO/Glass substrate	1.00	35.1	0	13.23	−74.56
S2	ZnO NRs without seed layer	2.58	76.2	0	61.18	−26.62
S3	ZnO NRs with seed layer	27.55	76.2	0	151.94	64.14

Note: The ${\theta }_{\mathrm{A}}$ values listed for S2 and S3 represent the intrinsic water contact angle measured on a flat ZnO surface, which reflects the intrinsic surface chemistry required by the thermodynamic model, distinct from the apparent contact angles measured on the actual nanostructured surfaces in Figure 5. The ${\theta }_{\mathrm{B}}$ values represent experimental contact angles on flat reference substrates, confirming complete wetting by the silicone oil.

The enhanced stability of S3 is attributed to the densely packed and vertically aligned ZnO nanorods, which promote robust capillary confinement of the lubricant. Its high roughness factor (R = 27.55) reflects the substantially increased surface area available for lubricant retention. Because conventional AFM under-represents the topography of dense, high-aspect-ratio nanostructures [27], the roughness factor of S3 was calculated using a simplified geometric model derived from FE-SEM observations. Conversely, the roughness factor of S2 was determined directly from AFM data with representative topographies provided in Figure S1. Finally, the positive ΔE₂ value and the preserved droplet mobility after the comparative durability test support the superior lubricant stability and dynamic repellency of S3.

Figure 7 presents the droplet velocity on SLIPSs before and after the comparative durability test using a spin coater at various rotational speeds. Each condition comprised four 1-min spin cycles to assess degradation in slippery performance. For samples subjected to 1000 rpm, the sliding velocity decreased from 0.68 ± 0.05 to 0.58 ± 0.02 mm·s⁻¹, representing a ~14.9% reduction. This relative stability indicates the effective retention of lubricant within the nanostructured matrix at the lower rotational speed. Higher rotational speeds accelerated lubricant depletion; spinning at 1500 rpm reduced velocities from 0.74 ± 0.05 to 0.49 ± 0.03 mm·s⁻¹ (a 33.3% decrease), while spinning at 2500 rpm induced a 38.4% drop from 0.67 ± 0.06 to 0.42 ± 0.02 mm·s⁻¹. These findings indicate that increasing the rotational speed promotes lubricant depletion, leading to a proportional loss of surface slipperiness.

Figure 7. Droplet velocity on SLIPSs after repeated 1-min spin-coating cycles at various rotational speeds.

The reduction in droplet velocity is attributed to the thinning or partial depletion of the lubricant layer on the ZnO nanostructured surface [28]. Higher rotational speeds may promote lubricant depletion, thereby enhancing contact-line pinning and droplet adhesion, consequently reducing droplet mobility. These results highlight the critical need to maintain lubricant stability for durable SLIPS performance [29].

3.3. Thermodynamic Analysis of Bacterial Adhesion and Anti-Biofouling Performance

Figure 8 shows confocal laser scanning microscopy (CLSM) images of E. coli adhered to the sample surface after bacterial incubation, providing a qualitative visualization of bacterial adhesion behavior. The cells exhibit a characteristic rod-like morphology, measuring approximately 1–3 µm in length and 0.6–1.0 µm in width, which is consistent with typical E. coli dimensions [30]. The overview reveals a non-uniform spatial distribution with variations in cell density across the surface. At higher magnification, the bacteria form localized clusters rather than uniformly dispersing as isolated individuals. Such early-stage clustering is commonly associated with the initial phases of bacterial colonization and may represent early-stage biofilm development under prolonged exposure [31,32]. A quantitative evaluation of bacterial surface coverage via fluorescence-based analysis is detailed below.

Figure 8. Confocal laser scanning microscopy (CLSM) images of E. coli on the sample surface: (a) overview; (b) higher-magnification view.

To verify that the fluorescence signals detected in subsequent CLSM imaging originated from bacterial cells rather than background emission from the surface materials, the intrinsic optical responses of the slippery liquid-infused nanostructured surface were examined. Figure 9 presents the UV–Vis absorption and photoluminescence (PL) spectra of ZnO nanorods and silicone oil, representing the structural and liquid components of the slippery surface. As shown in Figure 9a, the absorption spectrum of ZnO nanorods exhibited a distinct peak at ~356 nm, corresponding to the intrinsic band-gap transition of ZnO. The corresponding PL spectrum displayed a characteristic emission peak at ~386 nm [33]. For the silicone oil, the UV–Vis spectrum exhibited an absorption peak at ~211 nm, while the PL spectrum exhibited an emission band at ~330 nm, as illustrated in Figure 9b [34,35].

Figure 9. UV–Vis absorption and photoluminescence (PL) spectra of the slippery liquid-infused nanostructured surface components: (a) ZnO nanorods (NRs) and (b) silicone oil.

These results indicate that the photoluminescence emissions of both ZnO nanorods and silicone oil occur predominantly in the ultraviolet region. Conversely, the intrinsic autofluorescence of E. coli typically occurs within the green spectral range (500–550 nm), which is driven primarily by endogenous biomolecules [36,37]. Consequently, the PL properties of the substrate components do not interfere with the bacterial fluorescence signals. This confirms that the obtained images accurately represent bacterial adhesion that is free from background interference from the underlying materials.

The anti-biofouling performance was evaluated by monitoring bacterial adhesion on bare ITO, ZnO nanorods, and SLIPSs after 1, 3, and 24 h incubation. Qualitative CLSM images are shown in Figure 10, and the corresponding quantitative surface coverage is analyzed in Figure 11. After 1 h, the initial attachment of individual E. coli cells appeared as sparse and isolated fluorescence signals across all substrates, yielding a negligible surface coverage of ~0.1%. However, a distinct divergence in colonization behavior emerged at 3 h; the density and distribution of bacterial signals increased substantially on the ITO and ZnO surfaces, reaching coverages of 35.9% and 45.0%, respectively, which were accompanied by the appearance of cell clusters. By 24 h, these clusters had developed into dense aggregates with surface coverage up to 50.8% on ITO and 54.6% on ZnO, indicating progressive bacterial colonization. Conversely, the SLIPS consistently exhibited minimal fluorescence throughout the 24 h period, maintaining a low coverage between 0.1% and 0.3%. These findings demonstrate its robust resistance to early bacterial adhesion and colonization. However, the present biological evaluation was limited to a single E. coli strain and an exposure period of up to 24 h. Therefore, the observed anti-biofouling behavior should be interpreted within these experimental conditions.

Figure 10. Fluorescence images of (a) ITO/glass substrates, (b) ZnO nanorods, and (c) slippery surface after exposure to E. coli for different durations. Green fluorescence indicates the presence of E. coli cells.

Figure 11. Bacterial surface coverage on ITO, ZnO nanorods and slippery surface after exposure to E. coli for different durations. Error bars represent the standard error calculated from three measurements at different locations on each specimen.

To further investigate bacterial adhesion, the surface energy components of the investigated substrates were determined using the van Oss–Chaudhury–Good (vOCG) approach combined with the Young–Dupré equation, which was expressed as follows [18]:

$${\mathsf{\gamma }}_{\mathrm{L}}\left(1+\mathit{cos}\mathsf{\theta }\right)=2\left[\sqrt{{\mathsf{\gamma }}_{\mathrm{S}}^{\mathrm{LW}}{\mathsf{\gamma }}_{\mathrm{L}}^{\mathrm{LW}}}+\sqrt{{\mathsf{\gamma }}_{\mathrm{S}}^{+}{\mathsf{\gamma }}_{\mathrm{L}}^{-}}+\sqrt{{\mathsf{\gamma }}_{\mathrm{S}}^{-}{\mathsf{\gamma }}_{\mathrm{L}}^{+}}\right]$$

(3)

where ${\mathsf{\gamma }}^{\mathrm{LW}}$, ${\mathsf{\gamma }}^{+}$ and ${\mathsf{\gamma }}^{-}$ are the Lifshitz–van der Waals, electron acceptor (acid), and electron donor (base) components of surface free energy, respectively, with subscripts S and L denoting the sample surfaces and probe liquids.

The vOCG approach has been previously applied to characterize the surface energetics of lubricant-infused surfaces based on apparent contact-angle measurements [38]. In this paper, the surface free energy components of the investigated substrates were determined by measuring contact angles with three probe liquids with known surface energy parameters as detailed in Table 1. For SLIPSs, these measurements represent the apparent wetting behavior of the lubricant-infused interface [39]. The resulting contact angles and calculated surface free energy values for the ITO, ZnO, and SLIPSs are summarized in Table 3. Both ITO and ZnO exhibited low contact angles across water, ethylene glycol, and diiodomethane, indicating hydrophilic behavior and high surface energy. Conversely, the SLIPSs showed a higher water contact angle of 101.51°, a moderate ethylene glycol contact angle of 76.87°, and a relatively low diiodomethane contact angle of 58.62°. These results suggest that the lubricant layer selectively repels polar liquids while maintaining partial wettability toward non-polar liquids.

Table 3. Contact angles and surface free energy components of the sample surfaces (W, water; E, ethylene glycol; D, diiodomethane).

Sample	Contact Angles (deg.)			Surface Free Energy (mJ·m−2)
	${\mathsf{\theta }}_{\mathbf{W}}$	${\mathsf{\theta }}_{\mathbf{E}}$	${\mathsf{\theta }}_{\mathbf{D}}$	${\mathsf{\gamma }}^{\mathbf{Tot}}$	${\mathsf{\gamma }}^{\mathbf{LW}}$	${\mathsf{\gamma }}^{\mathbf{AB}}$	${\mathsf{\gamma }}^{+}$	${\mathsf{\gamma }}^{-}$
ITO	35.05	41.08	39.91	98.052	39.655	58.397	58.276	0.121
ZnO	28.50	38.04	21.12	110.893	47.442	63.451	62.881	0.570
SLIPS	101.51	76.87	58.62	30.208	29.370	0.838	0.817	0.021

The calculated surface free energy values further elucidate this wetting behavior. The total surface energy of ITO and ZnO was 98.052 and 110.893 mJ·m⁻², respectively. Their electron-acceptor components (${\mathsf{\gamma }}^{+}$ = 58.276 and 62.881 mJ·m⁻²) were considerably higher than the electron-donor counterparts (${\mathsf{\gamma }}^{-}$ = 0.121 and 0.570 mJ·m⁻²). This indicates that these substrates favor acid–base interactions with Lewis base groups, specifically carboxyl, hydroxyl, and phosphate functional groups, that are present on the E. coli outer membrane, thereby explaining the high bacterial adhesion observed experimentally [40]. In contrast, the SLIPS exhibited a significantly lower effective total interfacial energy of 30.208 mJ·m⁻² with ${\mathsf{\gamma }}^{+}$ and ${\mathsf{\gamma }}^{-}$ reduced to 0.817 and 0.021 mJ·m⁻², respectively, reflecting the non-polar nature of the lubricant layer. These results indicate that the infused lubricant reduces the effective interfacial energy and polar interactions with bacterial cells, which may contribute to the observed reduction in E. coli surface coverage.

To evaluate the interfacial interaction strength governing bacterial attachment, the work of adhesion ($W_{\mathrm{SB}}$) was utilized as a primary comparative indicator. Specifically, the $W_{\mathrm{SB} }$between the sample surfaces and E. coli cells was calculated by integrating their respective surface energy components according to the following equation:

$$W_{\mathrm{SB}}=2\left[\sqrt{{\mathsf{\gamma }}_{\mathrm{S}}^{\mathrm{LW}}{\mathsf{\gamma }}_{\mathrm{B}}^{\mathrm{LW}}}+\sqrt{{\mathsf{\gamma }}_{\mathrm{S}}^{+}{\mathsf{\gamma }}_{\mathrm{B}}^{-}}+\sqrt{{\mathsf{\gamma }}_{\mathrm{S}}^{-}{\mathsf{\gamma }}_{\mathrm{B}}^{+}}\right]$$

(4)

where ${\mathsf{\gamma }}^{\mathrm{LW}}$, ${\mathsf{\gamma }}^{+}$ and ${\mathsf{\gamma }}^{-}$ are the Lifshitz–van der Waals, electron acceptor (acid), and electron donor (base) components of the surface free energy, respectively, with subscripts S and B denoting the sample surfaces and the bacterial surface.

To ensure consistency with experimental observations, the surface energy parameters for Escherichia coli (XL1-Blue), the specific strain utilized in the adhesion tests, were obtained from the literature [41]. The values used for the calculation were ${\mathsf{\gamma }}_{\mathrm{B}}^{\mathrm{LW} }$ = 35.0 mJ·m⁻², ${\mathsf{\gamma }}_{\mathrm{B}}^{+}$ = 1.7 mJ·m⁻², ${\mathsf{\gamma }}_{\mathrm{B}}^{- }$ = 55.1 mJ·m⁻². Based on these parameters, the calculated $W_{\mathrm{SB} }$ values for the ITO, ZnO, and SLIPS were 188.75, 201.19, and 77.92 mJ·m⁻², respectively, which is consistent with the bacterial coverages shown in Figure 11. The lower $W_{\mathrm{SB} }$ of the SLIPS suggests a reduced thermodynamic tendency for bacterial–surface interaction, which may contribute to the experimentally observed reduction in bacterial attachment. As multiple factors can influence bacterial adhesion, the calculated $W_{\mathrm{SB} }$values provide a comparative thermodynamic perspective on bacterial–surface interactions among the investigated surfaces. At the same time, other factors may also contribute to the observed bacterial attachment. Overall, these findings demonstrate the effective suppression of E. coli attachment by SLIPSs under the investigated conditions, where a stable fluid barrier prevents direct contact between bacteria and the underlying solid substrate.

4. Conclusions

ZnO nanorod-based slippery liquid-infused nanostructured surfaces (SLIPSs) were successfully fabricated on ITO/glass substrates via electrochemical deposition followed by PDMS-based silicone oil infusion. The incorporation of a ZnO seed layer promoted the growth of densely packed, vertically aligned nanorods, which significantly enhanced lubricant stability compared to non-seeded counterparts. Thermodynamic analysis demonstrated that only the seed-layer-assisted SLIPS satisfied the criterion for resistance to water-induced lubricant displacement, yielding a positive ΔE₂ value of 64.14 mJ·m⁻². This thermodynamic prediction was consistent with the retention of droplet mobility after the comparative durability test under rotational conditions. However, the decrease in sliding velocity indicated a decline in slippery performance.

Furthermore, anti-biofouling evaluation revealed that the SLIPSs effectively suppressed Escherichia coli attachment, maintaining bacterial surface coverage below 0.3% even after 24 h of incubation. In contrast, pristine ITO and ZnO nanorod surfaces exhibited substantially higher colonization. Surface free energy analysis showed that lubricant infusion reduced the calculated work of adhesion to 77.92 mJ·m⁻², which is consistent with the suppressed bacterial attachment. Ultimately, these findings provide insights into the interfacial factors associated with lubricant retention and bacterial adhesion, supporting the use of thermodynamic and interfacial energy frameworks to predict and evaluate SLIPS durability and anti-biofouling performance.