The chemical and physical interaction of a surface with a microbe is modulated to impart an antimicrobial nature to a surface. This involves tuning the surface through physical or chemical methods. To facilitate readability, the literature review on developing antimicrobial surfaces has been classified into four sections.
The above classification is based on the type of modifications/approaches used to develop an antimicrobial surface. The first strategy, i.e., patterned surface is a physical modification of surface in which microbes are either unable to come in contact with the surface due to steric hindrance or killed due to physical disruption of cells owing to penetration of the surface features. The second class is that of the functionalized surfaces where a purely chemical modification is done to either inhibit microbe–surface interaction or killing the microbes on interaction. The next approach is to produce superwetting surfaces which require a combination of both physical and chemical modification. It is known that for developing superhydrophobic and superhydrophilic surfaces both roughness (created by physical modification) and chemical modifications are required. However, these surfaces can only inhibit microbial adhesion and are thus treated as a separate category. It must be pointed out here that the above-discussed classes are maybe effective either in killing or inhibiting microbial adhesion. Recently, various surfaces with multiple modes of action have been sought such as surfaces with switchable properties which can both kill and inhibit the microbes simultaneously. These types of surfaces can be developed by incorporating biocidal agents and stimuli-responsive polymers as antifouling material. These approaches have been summarized as the fourth category of smart surfaces, the schematic of all the strategies is illustrated in Fig. 2.
Nature is full of examples including lotus leaf, taro leaf, and shark skin that prevent microbial attachment as a result of micro and nanostructured surfaces. Some surfaces for instance gecko skin, cicada wings, and dragonfly wings can even exhibit microbicidal activity . Details of the structure and activity of some of the natural biocidal surfaces are shown in Table 1.
The biocidal efficiency of a patterned antimicrobial surface is dependent on the surface topography and the microbe species. Hasan et al. have studied the antibacterial action of the cicada wing surface on both gram-positive and gram-negative bacteria of rod-shaped and coccoid shape. The surface topography of cicada wings consists of hexagonally packed uniform nanopillars of height 200 nm, base and cap diameter of 100 nm and 60 nm, respectively, and spacing of 170 nm for Psaltoda claripennis species (of cicada). It was found that only gram-negative bacteria, regardless of their shape, are significantly distorted and eventually killed by the wings of cicada . This is because the cell walls of both gram-positive and gram-negative bacteria are different. The cell wall of gram-positive bacteria comprises several layers of peptidoglycan which makes it more rigid compared to that of gram-negative bacteria that have a single layer of peptidoglycan and a second layer consisting of phospholipids.
Likewise, Yang et al. have reported antibacterial surfaces by developing a honeycomb pattern on a silicon wafer with varied pore sizes from 0.5 to 10 µm using photolithography and deep reactive ion etching. It was observed that the pattern of 1 µm significantly decreases the bacterial adhesion and growth and biofilm formation of S. aureus and E. coli. The reason behind the enhanced antibacterial behavior of 1 µm pattern size is due to the two key aspects. First is the accessibility of favorable attachment sites. The attachment sites must facilitate a greater contact area between cell and substrate with minimal cell damage. For 1 µm pattern, preferable adhesion sites are much lesser as compared to that of larger pattern size, as 1 µm pattern causes large cell deformation by trapping the cells inside the pores. This was observed for E. coli cells which have a similar dimension as that of the pattern. The second aspect is the physical confinement which hinders the growth and proliferation of bacteria . A similar result was seen for micron-sized patterns of comparable dimensions . Xiang et al. have reported the effect of micro-nanopillar array on bacterial inhibition. Titania (TiO2) micro-nanopillar with motif size of 0.6 µm reduced bacterial adhesion by 62% for S. aureus (cell size is 1 µm) and 73% for E. coli (cell size is 1–2 µm long and 0.5 µm wide) after 30 min as compared to that of flat surface . Similarly, Ivanova et al. have reported the bactericidal activity of patterned black silicon. According to their findings, 500 nm height nanoprotrusions showed similar killing efficiency to that of dragonfly wings for S. aureus and B. subtilis with a killing rate of 4.5 X 105 cells killed cm−2 min−1 and 1.4 X 105 cells killed cm−2 min−1, respectively. However, black silicon showed higher killing efficiency than dragonfly wings for P. aeruginosa .
Rosenzweig et al. have developed a poly (methyl methacrylate) nanopillar structure and studied their fungicidal properties. The nanostructured surface pillars were fabricated with a periodicity of 170, 320, and 500 nm. It was observed that nanopillar structure showed antifungal activity in the increasing order of periodicity 170 nm < 320 nm < 500 nm as shown in Fig. 3 (a) and (b). Compared to the flat surface, spores started deforming on the nanopillared surface and the development of the germ tube is disrupted and detachment of spores starts after 16 h and 8 h for A. fumigatus and F. oxysporum, respectively. This result may appear contradictory to the observations for antibacterial activity discussed above as the sizes of the cell and mechanosensing mechanism are different [51, 52]. However, the fundamental understanding behind the mechanism of antimicrobial activity of patterned surfaces is very rudimentary at this stage and needs further work.
Furthermore, Hasan et al. have reported antibacterial and antiviral Al 6063 alloy nanostructured surface prepared by wet chemical etching. The nanostructures of width 23 nm ± 2 nm were orientated randomly into parallel ridges with a root-mean-squared roughness of 995 ± 114.7 nm. To check the effect of surface structure on antibacterial properties, S. aureus (gram-positive) and P. aeruginosa (gram-negative) bacteria were tested. Both types of bacterial cells were deformed on the nanostructured surface and more than 87% of attached cells were rendered nonviable. The reason for bactericidal activity was attributed to the rupturing of the bacterial cells. The effect of nanostructured surfaces on viruses was studied using Respiratory syncytial virus (RSV) and Rhinovirus (RV). Within two hours of exposure, the etched Al surface showed remarkably lower viable viruses compared to that of flat Al surface and all viruses were killed after 24 h. However, the RV virus was more susceptible to the nanostructured surface as compared to that of the RSV virus. Also, the same surface was tested against SARS-CoV-2, and virus viability was examined for different intervals of time up to 48 h. The viability of viruses was reduced after three hours of exposure, and no live viruses were observed after six hours of exposure. In comparison, on the control surface, significant depletion of live viruses was noticed only after 24 h of exposure. A period of 48 h is needed for the elimination of all the viable viruses from the control surface. The reason for virucidal activity is not clear, and the authors have attributed it to nanoscale roughness which can rupture the virus envelope. Also, the size of the viruses is very small and thus can get trapped in the structure which may be detrimental to their viability [53, 54].
From the above literature of biocidal activity on patterned surfaces, we can conclude that the biocidal mechanism on the patterned surface is due to the cell rupturing upon penetration of surface followed by the death of microbes as shown in Fig. 4. However, this mode of action may not be equally effective against the microbes having thicker cell walls or additional envelopes. Another important reason for the antimicrobial activity is the trapping of cells in the structure leading to cell death as shown in Fig. 5. The summary of the literature on patterned antimicrobial surfaces is included in Table 2.
Functionalized surfaces can be developed by modifying a surface with a material that can actively kill or inhibit the microbes. The mode of action for functionalized surfaces can be through contact killing of the microbe by functionalized surface due to the materials chemical groups , or the functionalized surface generates heat, reactive species on exposure to external stimuli to disrupt the activity of microbes .
The chemically active functionalized surfaces involve the use of non-leachable materials such as polycations, which provide effective biocidal activity through direct contact with microbes. Such a surface with polycation functionalization enhances the adsorption of negative surface charged microbes by electrostatic interaction between the cell membrane and material surfaces . As a consequence, the genetic material of the microbes undergoes leakage and loss its effectivity. This is one of the most promising approaches for a wide range of microbes.
Lin et al. have reported the role of polyethyleneimine and its molecular weight on bactericidal (S. epidermidis, S. aureus, P. aeruginosa, and E. coli) and fungicidal (S. cerevisiae, C. albicans) activity. They observed that higher molecular weight polymer showed higher microbicidal activity. This is because the polymer chain length higher than or equal to the size of bacteria facilitates easy penetration into the bacterial cell and destroys the cell membrane .
Wong et al. have reported layer-by-layer film of N,N-dodecyl,methyl-polyethylenimine with a polyanion, such as poly(acrylic acid) to develop an effective microbicidal surface against S. aureus, E. coli, and influenza (H1N1) virus. Both high positive charge density and length of alkyl chains are important parameters. Also, a higher number of bilayer deposition was necessary for higher virucidal activity as compared to that of bacteria, as the size of a virus (~100 nm) is approximately 1/10th size of bacterium (~1 µm). The lower antiviral activity observed for a lesser number of bilayers is due to the fact that the voids which are present on the surface are too big to fit a virus. The possible mechanism for antimicrobial activity is through contact killing of microbes by polycationic chains .
Silva et al. have developed Silica NPs modified surface using positively charged amine group which resulted in a 50% reduction of vesicular stomatitis virus G (VSV-G) transduction. This is due to the strong interaction of modified silica particles with the virus, which resulted in the blockage of direct contact among cells and viral particles . Likewise, Meder et al. have developed colloidal alumina particles functionalized with an amine group and different functional groups to investigate their controlled interaction with viruses .
Donskyi et al. have shown the antiviral activity of functionalized nanographene sheets through synergistic electrostatic and hydrophobic interaction. They have developed nanographene derivatives functionalized with polyglycerol sulfate and fatty amine and investigated their attachment with the HSV-1 virus. Polyglycerol sulfate allows electrostatic interaction with the virus, while alkyl chains provide enhanced antiviral action through hydrophobic interactions .
It has been observed that some surfaces are effective against only enveloped viruses but not against non-enveloped viruses. Tuladhar et al. have reported the role of hyperbranched quaternary ammonium coating against influenza virus (enveloped) and poliovirus (non-enveloped) virus. Virucidal activity of hyperbranched quaternary ammonium was found to be effective for influenza virus alone and the mode of action is through the disruption or detachment of viral envelope by the long-chain lipophilic tails and the high-density end groups .
Thus, functionalized surfaces are active against a vast spectrum of gram-positive and gram-negative bacteria, viruses, and fungi. The factors responsible for biocidal activity are the chain length of the alkyl polycation, molecular weight of the polymer, charge density, and so on. For instance, the highest activity against gram-positive bacteria and yeast was shown by a QAC containing chain length of 12–14 carbons, whereas chain length with 14–16 carbons shows the highest activity against gram-negative bacteria [67, 68].
Other than chemically active functionalization, antimicrobial surfaces can be activated by physical methods. One such example is photothermal therapy. The mode of action is based on the generation of local heat by a photothermal agent upon exposure to light of a suitable wavelength. This results in hyperthermia and leads to microbial death by protein denaturation, rupturing of the cell membrane, cellular fluid evaporation, etc. [69, 70]. Yang et al. reported excellent photothermal activity of Silver functionalized SnS2 surface. Antibacterial activity of 100% growth inhibition against E. coli and S. aureus was achieved with an Ag-SnS2 concentration of 0.5 mg/mL after exposure to near-infrared (NIR) for 5 min. The sample was also tested for in vivo antibacterial activity against S. aureus on mice as shown in Fig. 6 . Similarly, photothermal antifungal and antibacterial activity was reported by Lei et al. Polydopamine nanocoating achieved 84%, 96%, and 93% killing efficiency for E. coli, S. aureus, and C. albicans, respectively, upon exposure to NIR . In addition, UV is also used to activate metal oxides such as ZnO and TiO2 that leads to the degradation of microorganisms via photocatalysis. Semiconducting oxide generates high-energy electron–hole pair when exposed to radiation (typically UV–visible light) with energy more than the bandgap. These high electron–hole pairs bring about redox reactions at the surface of the oxide particles, resulting in the generation of various reactive oxygen species and free radicals which induce oxidative stress in the microbial cell and lead to their death [73, 74]. Kim et al. have reported photocatalytic viral inactivation against MS2 bacteriophage, influenza virus, and murine norovirus using TiO2 nanoparticles prepared with different calcination temperatures. The sample calcined at 700 °C showed enhanced virucidal activity due to the presence of a mixed anatase–rutile phase .
The summary of various studies reported on antimicrobial activity of the functionalized surfaces is listed in Table 3.
The development of biofilm on an external surface occurs by the attachment of microbes to the surface. The crucial step to control biofilm formation is by developing antiadhesion surfaces that can restrict the contact between microbes and the surfaces. Such surfaces can be developed by enhancing the wettability of a material. This can be done by combining roughness with chemical treatment onto the surface . A surface is said to be superhydrophobic when it exhibits a water contact angle (WCA) above 150°. On such surfaces, water droplet rolls off even at a small tilting angle , whereas, superhydrophilic surface shows a WCA equal to or less than 5° and water droplet completely spread on the surface .
Superhydrophobic surfaces are efficacious for a wide array of microbes inclusive of various species of bacteria, fungus, and viruses. The superhydrophobicity imparts antimicrobial character by minimizing the attachment of microbes on the surface .
In a study where a lotus leaf-like surface made of Ti was reported, it was observed that the bacteria were unable to adhere to the superhydrophobic surface due to the presence of trapped air nanobubbles and microbubbles in the hierarchical nanostructure. The bacteria start to assemble at the tri-phase interface, as illustrated in Fig. 7. The nanostructure and the trapped air (hydrodynamic force) minimize the area of contact among bacteria and the surfaces imparting an antifouling activity .
Ellinas et al. have reported antibacterial superhydrophobic micro-nanotextured surface of poly (methyl methacrylate) (PMMA) having WCA greater than 155°. The surface exhibited high bacterial repulsion against cyanobacteria Synechococcus sp. for a long period of 72 h and low bacterial adhesion upon immersion in bacterial cell solution till the fourth day. It had been previously reported that the superhydrophobicity of a surface vanishes, when kept immersed, with time due to the eventual depletion of the air layer and leads to bacterial adhesion. This paper shows that the surface design and feature sizes are important parameters to avoid this depletion of the air layer and to maintain a superhydrophobic character . Privett et al. prepared an antifouling superhydrophobic surface using silica-colloid-doped fluorinated substrates. According to their findings, the presence of both roughness and chemical modification using low-energy materials are responsible for minimizing bacterial adhesion .
Yeongae Kim et al. have reported antifungal activity of superhydrophobic aluminum surface against Penicillium, Cladosporium, and Aspergillus which exhibited a water contact angle of 169°. They have observed that only superhydrophobic surface was not contaminated, while the superhydrophilic and hydrophobic surfaces were contaminated on the direct inoculation of fungal spores .
Like bacteria and fungus, superhydrophobic surfaces also show a reduction in viral adhesion. Katoh et al. have reported that the fabric of personal protective equipment made of non-woven polypropylene with enhanced WCA can potentially reduce the risk of virus carryover by repelling the infectious body fluids [89, 90]. The adhesion of infected fluid on PPE can lead to a higher risk of spreading infection. Hence, superhydrophobic surfaces can lower the risk of viral infection.
The antimicrobial mechanism of superhydrophobic surfaces is mostly restricted to avoiding microbial adhesion rather than actively killing them. One of the ways to enhance the antimicrobial activity of superhydrophobic surfaces is by combining the superhydrophobicity with antimicrobial metal or metal oxide nanoparticles. This alters the roughness and also actively kills the microbes present on the surface [91, 92]. Berendjchi et al. have reported superhydrophobic and antibacterial cotton surfaces using copper doped silica nanoparticles followed by hydrophobic modification with hexadecyltrimethoxysilane. The addition of copper (Cu) on the silica sol provides roughness to the silica surface and enhances the antibacterial property . Likewise, Singh et al. have reported antimicrobial white cement composite embedded with different amounts of zinc oxide (ZnO) nanoneedles. The surface showed biocidal action for E. coli (gram-positive bacteria), Bacillus subtilis (gram-negative bacteria), and Aspergillus niger (fungus) . Dimitrakellis et al. have reported bacteria repelling and bactericidal surface against E. coli developed by superhydrophobic micro-nanotextured PMMA surface modified with Cu (shown in Fig. 8). The hierarchical rough surface was developed by plasma etching of PMMA, and the biocidal activity was speculated to be due to its surface structure that allowing the mechanical killing of bacteria. The presence of superhydrophobic surface deposited by fluorocarbon (CFx) acted as a bacteria repealing surface by restricting the contact between bacteria and the surface. Finally, the Cu acted as a biocidal material and instantaneously killing any bacteria adhering to the surface . Similarly, other metals and metal oxides such as silver and titanium dioxide (TiO2) are also effective antimicrobial agents [94, 95]. The uptake of silver and copper ions or nanoparticles by microbes leads to their death by disrupting various cellular processes such as enzyme activity, DNA replication.
The water droplets on superhydrophilic surfaces spread promptly to completely wet the exposed area and make a compact water layer. This works as a barrier that restricts contamination on the exposed area and enables self-cleaning . Based on this property, superhydrophilic surfaces exhibit antifouling activity .
Qian et al. developed a superhydrophilic surface with antibacterial and antifungal properties on stainless steel surfaces by depositing polydopamine (PDA) and silver nanoparticles (AgNPs). This work was inspired by mussels and was further modified with a hydrophilic material methoxy-polyethylene-glycol thiol (mPEG-SH). The water contact angle achieved was close to 0°, and this superhydrophillic surface showed antibacterial and antifungal activity against S. aureus, E. coli, and Penicillium F2-1. The antibacterial and antifungal activities were due to the combined effects of the antiadhesion created by water layers, the bacteria-killing ability of AgNPs, and the stereo hindrance caused by mPEG-SH molecular chains as shown in Fig. 9. Upon immersion of the surface on the inoculated medium, a huge quantity of Ag ions discharged on the medium which leads to the killing of attached and neighboring bacteria. However, the antifungal properties of superhydrophobic surfaces were tested in presence of a humid atmosphere where the bound water layer was in direct contact with the humid air. Here the released Ag ions were present in the bound water layer which leads to antifungal activity. This water layer remains stable due to the strong attraction of superhydrophilic surface to the water, which helps in sustaining the antifungal property of the surface. Further, the incorporation of antimicrobial metal or metal oxides enhances the antimicrobial properties of superhydrophilic surfaces by allowing higher wetting [97, 98]. Other than metal or metal oxides, hydrophilic polymers are utilized for antimicrobial surfaces [99,100,101]. Polyethylene glycol (PEG) is one such polymer that shows enhanced hydrophilicity with stereo hindrance effect due to long-chain PEG [101, 102].
The summary of studies reported on the antimicrobial activity of various superhydrophobic and superhydrophilic surfaces is shown in Table 4 which indicates that like superhydrophobic surfaces, these surfaces also inhibit the adhesion of microbes mainly bacteria and fungus rather than killing them directly. The affinity of water with hydrophilic surface is more in the case of high surface energy material than that of organic molecules due to which water molecules bound tightly with the superhydrophilic surface and prohibited the cell surface interaction .
Various types of antimicrobial surfaces have been developed in the past few decades based on the strategies discussed above. However, surfaces with single functionality have some serious drawbacks such as antifouling surfaces cannot maintain their non-adhesive property for the long term and eventually leads to biofilm formation. Also, biocidal surfaces can effectively kill the microbes, but after a certain period of time, a higher amount of debris or dead microbes starts accumulating on the surface, and as a result, it affects the functionality of the surface . Therefore, combinational surfaces with kill and release ability can help in developing effective antimicrobial surfaces for the long term. The mode of action for developing smart surfaces is based on killing the microbes attached to the surface using various antimicrobial agents, and the dead microbes are released using stimuli-responsive polymers [11, 12, 108].
One such example is of thermoresponsive polymers such as poly(N-isopropylacrylamide) (PNIPAAm) which has its lower critical solution temperature (LCST) at 32 °C. Lopez et al. have reported dual functional antibacterial and antifouling surfaces against E. coli and S. epidermidis as shown in Fig. 10. In their works, lysozyme and quaternary ammonium salt (QAS) was used as a biocide and PNIPAAm was used as an antifouling material. The switchable surface was obtained by changing the temperature across the LCST of PNIPAAm. E. coli attach to the surface at 37 °C. An increase in the temperature above the LCST (37 °C) results in the collapse of the secondary chain structure of PNIPAAm. This exposes the underlying biocide which kills the bacteria. Also, as the temperature changed below the LCST of PNIPAAm (25 °C), the dead bacteria are released from the surface due to the conformational change of PNIPAAm [109, 110].
Similarly, Yan et al. have reported switchable surfaces consisting of an inner antimicrobial peptide (AMP) layer surrounded by a pH-responsive poly (methacrylic acid) (PMAA) layer as shown in Fig. 11. PMAA initially restricts bacterial adhesion due to its hierarchical surface. Once the bacteria colony formation starts, it increases the acidification of the surface which results in the collapsing of PMAA layer. This leads to the exposure of the AMP layer that kills the bacteria. In addition, the dead bacteria release from the surface as the hydrophilicity of the polymer resume due to an increase in the environment pH . In addition, Jiang et al. have reported smart surfaces with switchable antimicrobial and antifouling properties. They have developed a surface modified with poly(N,N-dimethyl-N-(ethoxycarbonylmethyl)-N-[2’-(methacryloyloxy)ethyl]-ammonium bromide) which showed bactericidal activity against E. coli by killing more than 99.9% E. coli in one hour. However, these surfaces act as an antifouling surface upon hydrolysis to form a zwitterionic polymer and released 98% dead bacteria .
Developing a biocide free surface for killing bacteria is of great interest as it does not have toxicity issues or a chance of evolving multidrug resistant bacteria. Photothermal agents (PTA) are one such example of biocide free materials where biocidal activity is due to the heat generated by the PTA upon exposure to light which results in bacterial cell damage . Qian et al. have developed switchable surfaces with bacteria killing and releasing ability using tannic acid/Fe3+ (TA/Fe) ion complex as a photothermal bactericidal agent and PNIPAAm as an antifouling material. TA/Fe showed biocidal activity against E. coli and methicillin resistant Staphylococcus aureus (MRSA) on exposure to NIR and all the dead bacteria were released from the surface at a temperature lower than the LCST of PNIPAAm . Similarly, another switchable surface with photothermal bactericidal activity has been developed by Qian et al. In this work, instead of using stimuli responsive polymer for switching the activities they have used gold nanoparticle layer and phase-transitioned lysozyme film (GNPL-PTNF). GNPL upon exposure to NIR showed bactericidal activity against E. coli and S. aureus with killing efficiency of > 99% and > 96% respectively, whereas PTNF on contact with vitamin C solution degraded and removed all the dead bacteria present on the surface .
However, these types of surfaces are mostly reported for bacteria. Some of the reports on smart surfaces with dual functionality are listed in Table 5.