Engineering a dual functional coating for shielding surfaces from bacteria and viruses
Our coating consists of two nanostructured components providing a pathogen repellent and microbicidal function, respectively (Fig 1, SI Note 1 and 2). Contamination by pathogen contained in liquid environments, such as respiratory droplets, is prevented by a water repellent superhydrophobic layer of fluorinated silica nanoparticles, robustly anchored on an underlying interpenetrated polymer network (IPN) film (see Materials and Methods and Note 3 in SI).
Effective microbicidal action is imparted by the incorporation of nanoscale grains of Zeolitic Imidazolate Framework-8 (ZIF-8) that act as a localized stimuli-responsive source of Zn ions (Fig 1b, SI Note 2). ZIF-8 is a zinc-based metal organic framework (MOF), resembling the zeolite topology, with 2-methylimidazole (HmIm) as the linker 22. The absence of hydrophilic functional groups in the HmIm makes ZIF-8 inherently hydrophobic, contributing to the coating superhydrophobicity 23. The spontaneous release of Zn2+ ions from ZIF-8, due to its gradual decomposition in a broad range of pH values bears promise for its use as an antimicrobial agent 24, 25. Here, the presence of ZIF-8 is aimed at killing bacteria that may adhere to the coating due to damage to its structural integrity, and thus localized loss of super-hydrophobicity, evaporation of their liquid shell environment before removal from the surface, or loss of the air layer due to prolonged immersion in a contaminated liquid (Fig 1b). For further discussion on the surface protection mechanism please see Note 5 in SI.
Protecting surfaces from viral contamination
We first explored the performance of our water repellent surface structure in avoiding viral contamination. We used a steel rod with significant surface roughness (Fig S15) and tested its ability to mediate transmission of viruses in its uncoated state, or coated with our water repellent coatings, with or without ZIF-8 nanoparticles (referred to as water repellent and dual functional coatings, respectively; SI Note 4, Fig S15). For each test, the rod was dipped into a high titre solution of an EGFP-expressing lentiviral vector as a surrogate for infectious virus. The rod was dipped in a series of inert washing solutions (fresh Dulbecco's Modified Eagle's Medium (DMEM)) to determine the level of initial contamination and ease of cleaning (Fig 2a, Fig S15). Virus was quantified in the washing solutions by incubation on a monolayer of susceptible cells followed by fluorescence microscopy to detect and count infected cells (Fig 2b).
The first washing solution represents the capacity of the rod to resist viral contamination upon exposure to a high concentration lentiviral (i.e. human immunodeficiency virus) solution (1×107 TU/mL). Analysis of the first washing solution reveals a significant capability of the coatings to prevent viral contamination of the rod (p=0.05, Fig 2c). More than an 11-fold decrease in cell infection was observed from the bare rod (9.4% (± 0.7%)) to the ones coated with a water repellent surface (0.8% (± 0.3%)). The dual functional coating was also effective in preventing viral contamination, with a roughly 3-fold decrease in cell infection compared with bare steel. Remarkably, no virus was detected upon the first wash on the rod coated with the water repellent coating and only a very small amount of virus was detected for the dual functional coating, that was eliminated after the 2nd wash. The reduction in virus between the first two steps (contaminated surface to wash 1) for the dual functional coating was ca. 11-fold, after which detection of virus became sporadic. By contrast, the bare steel carried virus through all four wash steps showing a linear decline, with infectivity reducing by around a half with each successive wash. More details of the specific coating performance are provided in Note 4 in SI.
Bacterial Pathogen Shielding Performance and Mechanism
We used bacteria to evaluate the shielding performance against larger and heavier pathogens than virus, and to gain direct insights into the shielding kinetics of our coatings. Fig S16 shows a schematic representation of the initial interface between bacteria suspended in a liquid and the coating surface, and its time evolution. A continuous air film, denoted as plastron layer, is initially formed at the solid-liquid interface preventing contact between bacteria and the coating surface. Over time parts of the plastron layer are removed via the formation and release of air bubbles, induced by the liquid pressure above the coating. These newly immersed coating surface becomes available for bacteria adhesion and eventually result in the formation of a biofilm. To investigate the specific shielding kinetics of our multi-scale rough coating, a droplet of bacterial suspension, in its exponential growth phase, was placed and kept on the coating surface and on other non-coated materials (bare steel) used as a comparison (Materials and Methods in SI, Fig S 4(a)).
Fig 3(a,b) shows in-operando in-situ confocal microscope images of a green fluorescent, bacteria droplet suspension on a bare steel plate and a steel plate coated with the dual functional coating. On the bare steel surface, the bacteria immediately settle and start the surface adhesion process (Fig 3(b)). In stark contrast, on our coating the bacteria suspension keeps floating above the plastron layer (white horizontal line), without being able to reach the underlying surface 26.
To gain further insights on the surface colonization mechanism, a droplet of bacterial suspension was kept on a bare and a coated steel surface and the cell behaviour was tracked with a confocal laser scanning microscope (CLSM) (Movie 1-2, Materials and Methods in SI, Fig S4(a)). Bacteria possess hair-like structures on their cell wall, denoted as flagella, which aids them in exploring microscale topography and facilitates adhesion to a surface. On the bare steel surface, the bacteria displayed nanoscopic vibrations around an equilibrium position (Movie 1&2). This is characteristic of a surface-bound motility behaviour referred as swarming, which is usually observed upon bacterial adhesion to a surface 26. More information regarding mobility and subsequent characterisation is described in Materials and Methods in SI.
Fig 3(c-e) and Fig 3(g-i) show a time sequence of 15 min of CLSM tracking (Fig S4(a), Movie 1-2) highlighting the dynamic response of the bacteria droplet suspensions on the bare steel and dual functional coating. Adhered bacteria were identified by lack of displacement and are false coloured in yellow. The average motion of the bacteria on both the surfaces were quantified by their average displacement between each time point (Fig 3(f, j)). Notably, bacteria cells on the bare steel surface had significantly (p<0.001) lower average velocity of 4.09 (± 1.42) µm/min than those on the water repellent surface (9.66 (± 1.43) µm/min).
Biofilm formation on an abiotic surface, such as the bare steel here, starts with a reversible primary adhesion stage followed by an irreversible secondary adhesion stage. The primary stage is greatly influenced by hydrophobic interactions between the cell and the surface 27. Our in-situ analysis (Movie 1) indicates that several bacterial cells on the bare steel surface have already entered the secondary stage, showing no translational movement, and firmly adhering to the surface. In contrast, on the water repellent surface, no bacteria cells lacking motion could be identified, indicating prevention of the primary adhesion stage.
To attain a quantitative assessment of the effectiveness of our surface structure in preventing bacterial adhesion, bare and coated steel substrates were immersed in a bacterial culture and stamped on agar plates (Fig S5), with or without a prior washing step in Phosphate Buffered Saline (PBS). Fig 4(a,c) shows axio-observer microscopy of the agar plates stamped with the washed and the unwashed bare steel substrates. Notbly, both samples resulted in the formation of a square-shaped layer of green fluorescing bacterial colonies, too densely packed to be counted. In stark contrast, the agar plates stamped with the un-washed water repellent coatings show only 12 (± 5) colonies (Fig 4(b)). Stamping the water repellent coatings after washing resulted in very few bacterial colonies, in the range of 2 (± 2) (Fig 4(d)). We attribute the small number of bacterial colonies, transferred from the un-washed coatings to the agar plate, to loosely adhered bacteria micro/nanodroplets. This is confirmed by the significant decrease in bacteria colonies upon a gentle washing step (Fig 4(d)), indicating the absence of biofilm formation (more discussion in Materials and Methods in SI).
To further quantify the efficacy in preventing bacterial adhesion, serial dilution of the residual PBS used to wash the samples after exposure to bacteria in their log growth phase was undertaken (Fig S5). Fig 4(e) shows that the bare steel samples resulted in 2.1×105 (± 2.1×104) and 1.4×105 (± 3.1×104) CFUs/mL colonies for gram-negative and gram-positive bacteria strains, respectively. Our coating successfully prevented up to 99.85% and 99.94 % of the gram-negative and gram-positive bacteria strain growth (significantly different with a p-value of less than 0.01 in a one-way ANOVA test) with only 297 (± 56) and 89 (± 24) CFUs/mL, respectively. To validate the essential role of the water repellent texture, control samples consisting of the same polymer coating without the fluorosilica layer were also investigated. The latter resulted in slightly higher CFUs/mL values than the bare steel surface, confirming that the polymer layer does not contribute to a decrease in bacterial adhesion.
Role of micro-scale coating defects on pathogen adhesion
To determine the role of micro-scale coating defect sites on pathogen adhesion, the water repellent fluorosilica layer was removed in a quasi-elliptic area of 62.8 µm2, exposing the underlying steel surface. A droplet of bacterial suspension was kept over the defective region of the coating and its intact water repellent surrounding (Fig S4(a)). The interface between the microscale defect and the water repellent region was continuously in-situ imaged by CLSM. Fig 5(a) shows an orthogonal projection of the droplet-substrate region. Over the intact water repellent surface (region A), a plastron layer with an above floating cloud of bacteria is observed. The plastron layer disappears on the defective coating area. The latter area consists of two parts (B,C), corresponding to the border with the intact region and the bulk of the defected area, respectively.
Bacteria over regions A and B exhibit a swimming behaviour whereas in region C, bacteria encounter the surface, due to the lack of the plastron layer. Movies 3 & 4 show the time-lapse view from the image plane 1 and 2 in Fig 5(a). Fig 5(b,c) shows static top view images taken after 14 min on these areas. Analysis of the velocity profiles in regions A and B (Fig 5(d)) confirm that the bacteria float over the intact surface and over the interface with the defected area with average velocities between 10.9 (± 1.6) and 11.5 (± 2.3) µm/min, respectively. Whereas C has much smaller average values of 4.0 (± 1.6), in line with the analysis of the bare metal and water repellent surfaces above (Fig 3(i, m)). We also performed SEM analysis of such a defected area challenged with a bacterial culture for 10 min (Fig 5(e-g)). Bacteria began to colonise the defective area of the sample (Fig 5(f)), whereas there was no evidence of bacteria adhered to the neighbouring non-defective regions of the sample (Fig 5(g)). These results show that any microscale defects can act as a colonization site for bacteria. This may explain the observed few isolated colonies of bacteria observed on the agar plates stamped with the unwashed water repellent surfaces (Fig 4(b)).
Failure kinetic of the water repellent shielding mechanism
In addition to the presence of defects on the coating, the long-term stability of the plastron layer is a key factor to prevent bacteria and other pathogen adhesion to the surface. Previous studies report that the plastron layer on super-hydrophobic surfaces is lost within 1- 1.5 h of continuous immersion in water (Table S1). Here, to investigate the stability of the plastron layer on our surface structure, coated steel substrates were immersed in a liquid bacterial culture at a depth of 2.5 cm, and the water-surface interface was continuously imaged by CLSM over a period of 8 h (Fig S4).
Fig 6(a-h) show images with green fluorescent bacteria on agar plates stamped with the water repellent steel substrates as a function of their immersion time from 1 to 8 h. Notably, for up to 4 h of continuous immersion very few bacterial colonies are able to adhere to the substrate (Fig 6(a-b)). For exposures of 4 to 6 h, a rapidly increasing number of bacteria colonies was observed (Fig 6(c-e)). While longer time results in complete colonization of the bacteria-challenged area (Fig 6(f-h)). For the full characterization of the failure kinetics of the water repellent shielding mechanism please refer to Note 5 in SI.
To gain insights on the mechanism determining the longer stability of the plastron layer on our water repellent surfaces, in situ studies were conducted by immersing our coating in a column of deionized water coloured with rhodamine B (Fig 6(j-m), Fig S4(b), Movie 5). The collapsing of the plastron layer was captured by the formation of a series of sequential contact point with the surface structure. This process rapidly accelerates after 6 h where the formation of bubbles supported by the rough surface features is observed. Subsequently, the bubbles are released resulting in the wetting of the surface as highlighted by the dye accumulation and the correlated increase in fluorescence intensity (Fig 6(l, m)). This lifetime of the plastron layer is attributed to the multiscale surface roughness of our coatings (see Note 5 in SI for more characterisation and discussion)
Secondary surface shielding mechanism
Our coating provides protection against both virus and bacteria until failure of the plastron layer. However, despite providing a longer stability than previous reports on water-repellent surfaces (> 4 h of continuous submersion), we acknowledged that the eventual dissolution of the plastron layer limits the breath of applications, for instance, in marine environments, membrane systems, or bathrooms, where an antibiofouling surface that could tolerate extended submersion is required. To this end, we have exploited the antimicrobial and hydrophobic properties of ZIF-8 nanograins (Fig S11) to impart a secondary shielding mechanism to the water repellent coatings. For material property optimization and wetting performance of the dual functional coating with ZIF-8 please see Note 2 in SI.
The capability of this surface composition to eliminate bacteria proliferation even in the event of dissolution of the plastron layer was demonstrated by long-term immersion of coated and bare steel substrates in a 5 cm column of solution containing 104 - 105 bacteria cells/mL (Fig 6(n)). After 1 day of immersion, there was no statistical difference between the bare and coated substrates. However, after 5 days of immersion, all the ZIF-8 containing coatings revealed a significant reduction in the cell counts of 2-3 orders of magnitude. More specifically, while on the bare substrates the bacteria colonies increased from 6×103 (± 7.5×102) to 2.1×105 (± 7.5×104) with immersion time increasing from 1 to 5 days, on the dual functional coatings the bacteria colonies decreased from 6.1×103 (± 2.6×102), 5.4×103 (± 3.7×102), 6.7×103 (± 8.2×102) to 1.8×103 (± 8.4×102), 6.9×102 (± 2.1×102), 1.1×103 (± 2.6×102) in the same time interval for the 5, 10 and 15 wt% ZIF-8 content, respectively.
The antimicrobial effect of the ZIF-8 became more accentuated with increasing immersion time with bacteria colonies further decreasing on the dual functional coatings after 9 days of continuous immersion. At such prolonged interaction between bacteria and coated surfaces, the impact of the ZIF-8 content became measurable, with the 15 wt% ZIF-8 containing coatings showing the smallest number of colonies of 3×101 (± 1.8×101) versus the 1.6×102 (± 6.9×101) of the 5 wt% ZIF-8 containing coatings and the 5.9×105 (± 2.8×105) of the bare surfaces.
We further evaluated the efficacy of this antimicrobial mechanism to prevent bacteria adhesion in case of damage to the coating and subsequent loss of its water repellent functionality. A scratch was imparted on the pure fluorosilica water repellent coatings and the dual functional coatings with 15 wt% ZIF-8. A droplet containing bacteria culture in log phase was left on the defect (Fig S7), upon which the samples were washed with PBS and then incubated for 24 h. Subsequently the samples were stamped on agar plates, then incubated and analysed by microscopy (Fig 6(o-r)). The bare steel samples, used as controls, shows significant bacteria adhesion (Fig 6(o)). The non-scratched water repellent coatings had no bacterial adhesion (Fig 6(p)) in line with the plastron layer-based bacterial repulsion mechanism. The scratched water repellent coatings made of pure fluorosilica particles resulted in bacteria adhesion in proximity of the scratched region (Fig 5(q)). Notably, the scratched dual functional coatings containing ZIF-8 nanoparticles resulted in no bacteria adhesion despite the loss of their water repellent functionality (Fig 6(r)).