Plasma technology in antimicrobial surface engineering

Recent progress in materials engineering and polymers science has resulted in the last few decades in the development of many new materials with improved mechanical, chemical, and physical properties. One of the very fast-growing fields is the development of biomaterials with anti-bacterial behavior.^1,2 The interest in the area is determined by the possible negative impact of microorganisms on the performance of materials³ and human health-related effects.⁴ The strategy in biocidal materials has been shifted more and more from the change of the bulk composition to the development of new surfaces capable either to suppress bacterial attachment to the surface or to release specific chemical components with anti-bacterial effects.³ Antimicrobial coatings have received more and more interest due to the high risks and costs associated with the development of infections and a possible increase in antibiotic resistance. The main areas of biocidal materials usage are the medical and health care sector,^5–7 textile industry,^8,9 food packaging,^10,11 marine industry,^12,13 indoor air quality,¹⁴ etc. Among other areas, there is an increasing demand for antimicrobial coatings in biomedical applications.¹⁵ In health care, the function of therapeutic and diagnostic devices can be weakened due to the adhesion of proteins and bacteria during clinical operation in contact with physiological fluids. When bacteria spread and significantly colonized, bacteria adhere to the biomaterial surfaces. In the next step, they bind to the extracellular matrix and form biofilms. The biofilm protects bacteria to resist the immune system and antibiotics, which results in final infections.¹⁵ Infections from a hospital or health care are regarded as the major challenge for global health care,¹⁶ where antimicrobial coatings are key factors to prevent or limit the spread of infections, including the use of urinary catheters, prosthetic heart valves, central venous catheters, and other equipment.¹⁷ 

Historically, wet chemistry methods were developed and applied first for manufacturing anti-bacterial materials. The methods to produce antimicrobial coatings include dip coating,^18,19 layer-by-layer deposition,²⁰ electrochemical deposition,²¹ and many others. However, wet chemistry processing is often associated with the complexity of the technology, extensive use of solvents, dependence on the treated materials, and low scalability. On the other hand, plasma surface engineering is considered an alternative approach and has numerous advantages in comparison with conventional methods. Plasma, the fourth state of matter, is a gaseous mixture of electrons, ions, radicals, neutral species, etc., but it remains macroscopically neutral.²² Mostly, plasma is generated through ionizing a gas by supplying electrical energy to it. Correspondingly, the highly reactive species in plasma can be manipulated by adjusting the electric power input. Therefore, plasma can establish a controlled reactive physicochemical environment that is amenable to enabling a variety of plasma-based surface engineering methods for surface activation, coating deposition, and surface nano-structuring of virtually any solid material. Plasma surface engineering generally occurs on the very top layer of a surface within a few nanometers that can merely alter the surface properties of the treated material in a desirable way for the intended application without affecting its bulk characteristics.²³ This characteristic has made the utility of plasma methods attractive in antimicrobial surface engineering over the past time. Plasma processing is one kind of technique that uniquely features several advantages over traditional wet chemistry approaches in the field of antimicrobial material. Namely, the following key features bring advantages to plasma methods:²⁴ 

• Plasma surface engineering offers a variety of possibilities to process materials on the desired scales ranging from micro- over nano- to molecular and even atomic scale for fine-tuning of surface morphology and surface chemistry;

• Plasma-based approach is arguably a solvent-free technique, one of the most promising environmentally friendly and green methods;

• Plasma engineering is reliable, reproducible, relatively inexpensive, and energy-efficient;

• Plasma approach can be considered a universal method and applied to different materials such as metals, polymers, ceramics, and composite;

• Plasma processes can be scaled up to industrial production relatively straightforwardly.

The unique features of plasma methods allow defining appropriate plasma approaches to reach specific requirements for the development of new anti-bacterial materials.

This Tutorial aims to provide the readers with an introduction to the main plasma methods applied for the fabrication of antimicrobial surfaces. The method covered in the Tutorial ranges from low-pressure discharges to atmospheric pressure and aerosol-assisted plasmas. The Tutorial emphasis is made on the description of the backgrounds of physical processes taking place during the anti-bacterial surface engineering. Some typical applications in the field of biocidal materials are presented to illustrate the capability and flexibility of plasma methods to modify surfaces of metals, ceramics, polymers, and others.

The Tutorial is organized as follows:

• Section II provides an overview of different mechanisms defining the anti-bacterial activity of surfaces;

• Section III presents a comprehensive overview of different plasma-based methods for anti-bacterial surface engineering. The intention of the section is to give insights into the background, capabilities, and limitations of the methods. The methods of surface nano-pattering, surface etching, functionalization, and polymerization are presented and discussed;

• Conclusions and future outlooks in the field of plasma development of anti-bacterial coatings and surface engineering are given in Sec. IV.

The Tutorial is expected to provide the readers with the description of currently available plasma technologies in the field of anti-bacterial materials and helps with an appropriate choice of the methods for surface engineering to manufacture advanced materials with biocidal activity.

Currently, there are different approaches capable to help in reducing possible bacterial surface attachment and biofilms growth. As plasma-based methods are typically applied to modify the top layer of materials, here we will classify the surfaces based on the mechanism they provide to remove bacteria, suppress bacterial growth, or kill bacteria. As accepted in the literature, the three most general mechanisms are presented in Sec. II, which are

• Contact-killing, where the surface morphology directly affects the bacteria on nano- or micro-level physically damaging the structure of the microorganisms;

• Antifouling surface, where either morphology or chemistry or combination of both helps to prevent bacteria attachment to the surface;

• Drug-release surfaces, a large class of materials where the top surface is used to release specific compounds (metal atoms, ions, organic molecules, reactive oxygen and nitrogen species, etc.), capable to stop bacterial growth or killing the microorganisms.

Below a short overview of the above-mentioned classes of surfaces is discussed and principles of anti-bacterial mechanisms are presented.

Contact-active surfaces are capable of contact-killing anti-bacterial effects that exhibit microbial activity without releasing biocidal substances. Such surfaces can be created by fixing certain biocides onto them or a recently developed approach of surface nano-patterning.²⁵ The basic principle of microorganisms interaction with contact-killing surfaces is illustrated in Fig. 1(a),²⁶ whereas an approach of surface nano-patterning with the aim of bacterial destruction is presented in Fig. 1(b) for P. aeruginosa.²⁷ 

Among the mechanisms responsible for contact-active surface effects, the main mechanisms are believed to be

• a so-called spacer effect, where the biocidal group is attached to the surface through a polymer chain, allowing the biocide to reach the cytoplasmic membrane of the bacteria and to perforate them;²⁸ 

• positively charged quaternary ammonium compounds can detach phospholipids from the cell membrane and thereby kill the bacteria;²⁹ 

• nano-structures presented on the surface of material directly interact with bacteria resulting in mechanical penetration of the microorganism membrane and killing as demonstrated in Fig. 1(b) for diamond-like nano-structures.

It is important to emphasize that the mechanism of contact-active surfaces depends on the chemical composition of the surfaces or nano-scale morphology of the surface and no biocidal substance is released during the application. Correspondingly, any possible side effects of the contact-killing surfaces are minimized and the effect only lasts during the interaction of the material with bacteria. The method provides a possibility to engineer biomaterials with a high degree of biocompatibility and a low level of cytotoxicity. Very often the contact-killing effect, especially in the case of nano-patterned surfaces, is responsible for the first stage of the bacteria/anti-bacterial surface interaction when morphological features on the surface with a nano-size scale can provide mechanical damage to the bacterial membrane. In the following steps, the release of active anti-bacterial compounds takes place resulting in bacteria deactivation. Such effect is often attributed to Cu nano-structured surfaces.³⁰ Giannousi et al.³¹ demonstrated the anti-bacterial activity of copper-based nanoparticles and showed that copper nanoparticles (Cu NPs), as well as cuprous oxide (Cu₂O), induced plasmid DNA degradation. The effect was observed to be dose-dependent in Gram-positive and Gram-negative strains. Moreover, the authors showed that the concentration of released Cu²⁺ ions was below the level of inhibiting bacterial growth (minimum inhibitory concentration) and correspondingly was not the main parameter determining bactericidal effect. Based on these results, the authors concluded that the concentration of released ions was less important than the nanoparticle size for the anti-bacterial activity. In addition, as for the copper surface, it appears that the outer membrane would be the front line of bacterial defense against copper ions.³² Therefore, the size of Cu NPs turned out to be a major contributing factor for copper antimicrobial activity, which is realized through the contact-killing mechanism.

Surface coating with carbon nanotubes (CNTs), graphene, or diamond-like carbons (DLCs) promised interesting antimicrobial activity since these materials show relatively low cytotoxicity toward mammalian cells. The most frequently proposed mechanisms of action fall under different categories: including membrane damage because of direct mechanical puncture of the bacteria or mechanical stress on bacterial surface induced by interaction with sharp edges of nano-structures.³³ 

Most of the bacteria and other pathogen microorganisms not only colonize a surface by simple adhesion to a surface, but they can give rise to “biofouling.” Biofouling can be defined as the formation of a film composed of interacting microorganisms embedded in an extracellular polymeric matrix. In other words, this is an undesired but organized accumulation of biomacromolecules, such as proteins and other cellular products and microorganisms on a material.^34–36 

As illustrated in Fig. 2, the process of surface biofouling consists of different steps: (i) surface conditioning; (ii) initial bacterial adhesion; and (iii) biofilm formation. Hence, fouling starts with the adsorption of a layer of organic macromolecules (e.g., proteins and polysaccharides) on the surface acting as a conditioning film for bacterial adhesion.^34,37 It is important to consider that the following initial bacterial adhesion is partially reversible, and it can be more readily eradicated compared to the strongly adhering complex biofilm, formed in the following steps, through several reactions, including hydrophobic and electrostatic interactions. Indeed, initial bacteria-surface attachment and adhesion is the fundamental step for biofilm formation.

The settled biofilms in general have serious mechanical removal resistance, increasing the resistance to host immune responses and to antibiotic treatments compared to isolated microorganism colonies.

Biofouling has adverse effects in different technology fields: (i) on medical implants (e.g., prosthetic joints or catheters), biofouling causes device-related infections hardly responding to therapies, and necessitating revision surgeries; (ii) in food industry, biofouling is a main concern in equipment, cooling systems, and food storage; (iii) biofouling is a major concern also for air conditioning systems and boating.³⁸ 

Since the initial bacteria adhesion to the surface is more reversible than the settled biofilm, the main strategies to fight biofouling are oriented to prevent the initial bacterial adhesion, more than striking against the removal of the biofilms. This is, hence, carried out by engineered anti-adhesive coatings. Basically, two kinds of approaches can be considered, both working on extreme wettability (as reported in Fig. 3):

• Highly hydrophilic surfaces;

• Superhydrophobic surfaces.

In the first class, we can identify polyethylene glycol (PEG) and poly(2-methyl-2-oxazoline) (PMOXA), zwitterionic polymers, polysaccharides, and glycoproteins. Notwithstanding the different chemical structures, coatings based on these compounds have an antifouling activity due to a high affinity for water, in some cases forming a gel barrier, as mucin-like systems. In these cases, the hydrophilic chains react with water forming a hydration layer where contaminants, such as proteins, and bacteria can hardly adhere. This layer is a physical and energetic barrier to protein adsorption. Water molecules strongly solvate the non-fouling polymeric chains. To bind proteins to the surface, adsorbed water molecules must be expelled from the hydrated layer reducing free energy through solvation entropic effects. However, the energy balance of this process is not favorable: chain flexibility and packing, and surface wettability as well, play an important role in resisting protein adsorption due to an unfavorable decrease in entropy.³⁹ 

Such unique non-fouling properties have encouraged great efforts in the development of surface hydrophilization, mainly PEG-modified ones, through different approaches: covalent immobilization, physical adsorption, self-assembled monolayers, and plasma deposition.^40–48 

Superhydrophobic surfaces, inspired by the Lotus leaf, are characterized by a water contact angle higher than 150° and a slippery behavior: water is repelled by this kind of surface, and if contaminants are present on it, they are easily washed out.^49,50 Such surfaces can be artificially prepared by two approaches:

• Roughening of a hydrophobic surface;

• Roughening of a surface and the following functionalization with a hydrophobic coating.^51,52

In the case of superhydrophobic surfaces, the non-fouling activity against bacteria is mainly due to the very low surface energy that leads to reduced protein and bacteria adsorption. This is also related to the air layer entrapped between the protrusions of the rough surface, forming a barrier to the adhesion of contaminants having higher surface energy. On the other hand, possible adsorbed bacteria, and other contaminants, are easily removed due to the reduced adhesion force with the low surface energy material surface.⁵³ 

Although antifouling surfaces are greatly beneficial to reduce biofouling by preventing the initial bacterial adhesion to a surface, it is still challenging to completely eliminate bacteria adhesion, and some bacteria may still attach to a surface. Once bacteria adhere, as discussed above, a biofilm may develop on the surface and lead to more difficult treatment. This is the reason why multiple strategies are of the essence to combat bacterial infections.

As a supplementary of contact-killing and antifouling strategies, engineering surfaces with the capacity to release bactericidal drugs/agents is another well-known strategy for antimicrobial applications. Generally, drug-release surfaces are efficient to fight against the planktonic bacteria around a surface, newly attached bacteria to a surface, and even fully developed biofilms in some cases due to excellent bactericidal properties of released compounds.^15,54

Broadly, on the one hand, compounds with antimicrobial properties such as emerging antibiotics and metal (e.g., Ag, Au, and Cu) nanoparticles are commonly preloaded or embedded into a fabricated coating. In this way, these antimicrobial compounds are released with controlled kinetics from a fabricated polymeric matrix in some predefined fashion upon interaction with the operational environment and/or stimuli, e.g., pH, bacteria-secreted substances, and the above dual factors.⁵⁵ On the other hand, some coatings themselves can generate active bactericidal species (e.g., NO, H₂O₂, OH⁻, and singlet oxygen) in a desirable way through photoactivation and/or reaction with surrounding environmental substances, etc.^56,57

The main advantage of drug-release surfaces is in the controlled release of antimicrobial compounds to the local infected sites, which allows for delivering high doses of these compounds to the targeted loci, and thus reduces antibiotic resistance caused by long-term exposure to low concentrations of commonly used antimicrobial drugs.⁵⁸ In other words, drug-release coatings provide anti-bacterial activity only where needed, and they are thus highly suitable in some high requirement cases, e.g., the revision surgery after biomaterial-associated infection to clear the infection from surrounding tissues and local antibiotic prophylaxis in primary surgery.⁵⁹ 

However, given that a fabricated coating inherently acts as a reservoir for storing and releasing bactericidal drugs, antimicrobial drugs are easy to be depleted over time, ultimately leading to the surface being disabled. Therefore, the overall timeframe and kinetics of anti-bacterial actions should always be considered when designing such release-based antimicrobial surfaces for specific applications.

Figure 4 shows a schematic diagram of designing anti-bacterial coatings within a 4D perspective.¹⁵ In general, the release profile and release timeframe can be controlled by tailoring the properties of both the polymeric coating and the loaded drugs in it, through different wet chemistry methods [e.g., dip coating and conventional chemical vapor deposition (CVD)] and the emerging plasma-assisted approaches.^60–62 

In addition to the release kinetics of antimicrobial drugs that should maintain levels within a therapeutic window, sufficient to kill bacteria, the cytotoxicity toward normal cells or tissues (i.e., coating biocompatibility) must also be considered, which represents the current challenge in the field of engineering drug-release antimicrobial surfaces.

The fabrication of advanced nano-structured materials has become the heart of modern nano-science that facilitates many innovative applications including the focus of this Tutorial: anti-bacterial surfaces. Among many kinds of modern nano-fabrication techniques, non-thermal equilibrium plasma that uniquely features highly reactive species enables the precise control of surface morphology by a variety of plasma-based surface engineering approaches, e.g., plasma etching and plasma sputtering. Typically, these processes occur at low pressure but offer excellent flexibility in engineering surfaces with desirable bactericidal activities. Recent years have also witnessed advances in nano-patterning by atmospheric pressure plasma where nano-patterning is typically achieved either during plasma polymerization or chemical etching. Therefore, in this section, we will mainly focus on the low-pressure plasma etching, plasma sputtering, and the approach of aggregation cluster sources for nano-engineering. At the end of the section, a short introduction to atmospheric pressure nano-patterning will provide the readers with some examples of typical applications. The possibility of using these methods for fabricating anti-bacterial surfaces is also discussed in Subsection III.

Plasma etching that is known as a revolutionary extension of the physical sputtering technique is one of the most efficient approaches for surface nano-patterning. Historically, plasma etching was introduced to the field of integrated circuit manufacturing in the middle 1960s and became more popular in the early 1970s with the aim to reduce liquid waste disposal in manufacturing and achieve selectivity that were generally hard to reach with wet chemistry.⁶³ Now, it has become one of the cornerstones in modern semiconductor manufacturing and has also been widely extended for surface nano-patterning in multidisciplinary fields such as biology and energy.⁶⁴ 

In most cases, the term “plasma etching” is often synonymous with “reactive ion etching (RIE).” In general, both chemical and physical etching processes occur simultaneously in plasma etching, which makes it distinctive from traditional wet chemistry etching. Specifically, as plasma is partially ionized gas with an aggregation of ions, electrons, neutral species, excited particles, photons, etc., the reactive species in plasmas can react with the treated materials, which leads to consuming the material surface and forming volatile products. These volatile products can be further desorbed from the substrate and then extracted. Importantly, the energetic ions in plasmas can be accelerated directionally during the etching process by a sheath potential developed on surfaces exposed to the plasma, which enables a fast etching rate in the vertical direction than that in the sidewalls, making a perpendicular etching via ion bombardment on the treated surface.⁶³ The directional/anisotropic etching is one of the unique advantages of plasma etching over wet chemistry etching in which only isotropic etching occurs. Noticeably, the physical etching enabled by the direction-driven energetic ions can further promote the reactions between the reactive species and treated material, resulting in a high etching rate. This is another advantage of plasma etching over other techniques. The typical RIE process is presented in Fig. 5.⁶⁵ 

Since the RIE process mainly takes advantage of the high-energy particles in plasmas, the control over the etch rates, selectivity, sidewall profile, etch roughness, etc., can be achievable by tuning the processing parameters such as voltage, power, temperature, chamber pressure, and gas flow rate that directly influence ion density, electron temperature, and potentials of a plasma state.⁶⁶ In most cases, an appropriate selection of mask and gas plasma types is also necessary to obtain a desirable nano-patterning surface.⁶⁷ However, it is also possible to introduce well-designed nano-structures with maskless or self-mask plasma etching through various methods, e.g., using a reactive ions mixture of hydrogen bromide (HBr) and oxygen (O₂), or choosing different metals (e.g., Ag, Cu, Pt, and Si) to cover the reactor cathode that can enable the formation of passivating layers on the sidewalls of the etched profiles.^68,69 Overall, via operating the relevant parameters carefully, low-pressure plasma etching allows small scale etching (which can be as small as 10 nm) with extra advantages such as no contamination issues and no hazardous chemicals, while wet chemistry etching is inadequate to define small feature size less than 1 μm and also needs to handle hazardous chemicals and contamination issues with much more attention.⁷⁰ 

Regarding anti-bacterial surface engineering, it has been reported that some nano-structured surfaces can kill the attached bacteria.⁷¹ This is initially inspired by the bactericidal nano-pillar structures of cicada wings [Psaltoda claripennis shown in Fig. 6(a)].⁷² The subsequent mechanism studies have revealed that such a natural surface can kill bacteria solely by rupturing the bacterial cell membrane and not by chemical means [Fig. 6(b)].⁷³ Therefore, there is no need to worry about the chemical-induced cytotoxicity for such kind of anti-bacterial surfaces. Moreover, mechano-bactericidal biomaterials can efficiently avoid unintended issues such as the development of drug-resistant bacterial strains.

Following the work of discovering the fundamental bacterial mechanism of the natural insect wings (in addition to the Clanger cicada wings, examples such as dragonfly Diplacodes bipunctata wings also show the unique nano-structured bacterial contact-killing properties⁷⁴),⁷³ plenty of efforts have been devoted to the biomimetic mechano-responsive, anti-bacterial materials.^71,75,76 Plasma etching may play a constructive role in defining surface topographies for anti-bacterial purposes. For example, it has been reported that the RIE with SF₆ and O₂ plasma etched nano-pillar structures of black silicon [Figs. 7(a) and 7(b)] was highly bactericidal against all tested Gram-negative and Gram-positive bacteria and endospores (including P. aeruginosa, S. aureus, and B. subtilis), and exhibited estimated average killing rates of up to ∼450 000 cells min⁻¹ cm⁻².⁷⁴ Similarly, RIE with Cl₂/O₂ or O₂/SF₆ gas plasma-fabricated black silicon with nano-needle structures also showed excellent anti-bacterial properties for Gram-negative bacteria (E. coli), but Gram-positive bacteria (S. gordonii) were less unaffected by the nano-structured surfaces, likely on account of their smaller size, thicker cell membrane, and/or their lack of motility.⁷⁷ Therefore, the study may confirm the necessity to make the dimension of fabricated nano-structured surfaces consistent with the target bacteria. In addition to black silicon, materials like stainless steels and polymers that are widely used in biomedical fields are also etched by plasma etching approaches to impart them with bactericidal activities.^78,79 To be more specific, the study⁷⁸ demonstrates that Ar plasma etching with direct current (DC) discharge can lead to forming nano-pillars on AISI 316 stainless steel with a comparatively short processing duration. The plasma-fabricated nano-pillars can reduce the survival rate of both Gram-positive bacteria (S. epidermidis) and Gram-negative bacteria (E. coli). In the study,⁷⁹ the cone-like and pillar-like assays in nano- or micro-sizes were fabricated by Ar and/or O₂ plasma etching on polyetheretherketone (PEEK) surfaces [Figs. 7(c) and 7(d)]. Both the PEEK nano-arrays and micro-arrays can kill E. coli efficiently but in different mechanisms. The nano-arrays take advantage of the cell–surface interactions in stress and penetration which mimic the contact-killing mechanisms of nano-pillar surfaces of cicada wings, whereas the bactericidal activities of micro-assays could also be attributed to the enhanced adhesion induced by plasma treatments. As a result, a micro-assay can deform bacteria more effectively. Importantly, the plasma-fabricated polymer surfaces have also shown improved biocompatibility, exhibiting large potential in clinical applications.

On the other hand, different from contact-killing by nano-structured surfaces discussed above, some plasma etched nano-scale surfaces can also prevent the bacteria attachment by reducing the area available for contact, not by stressing mechanical killing.⁸⁰ Therefore, we have to admit that although there are a huge number of reports that have highlighted the role of nano-structures in bacteria attachment,^81,82 due to the difficult control over other parameters such as materials chemistries and bacterial strains that are also critical factors influencing bacteria attachment, the mechanisms behind bacteria attachment phenomenon remain unclear, which needs further effort to clarity in upcoming experiments.

Sputtering is a process of knocking atoms off a surface by bombardment with energetic particles, such as ions, atoms, molecules, or clusters of atoms/molecules. Using ions is the most straightforward approach from a technical point of view since charged particles can be easily manipulated by an electromagnetic field. Plasma can be adapted for sputtering because it is typically created by applying electromagnetic fields to gases (less often to liquids) and contains charged particles, including ions. The most straightforward implementation dates back to the 19th century when researchers investigated the physics of DC electrical discharges under low pressure using a diode configuration: a pair of metal electrodes placed opposite each other and sealed into a glass tube. It was noticed that over time, a deposit forms on the inner surface of the tube near the cathode, which, upon a more detailed study, turned out to be composed of the cathode material.⁸³ The phenomenon is based on the acceleration of positively charged ions from the plasma toward the negatively charged cathode. Typical values of the potential drop in the near-cathode region are hundreds of volts, and, therefore, ions arrive at the cathode surface with the corresponding energy. Such hundreds of eV energy are quite sufficient to break the surface integrity and knock out metal atoms from it. The sputtered atoms reach the adjacent surfaces, on which they form a thin film. As a result of ion bombardment, secondary electrons are also knocked out from the surface. They become accelerated by the electric field toward the anode, participating in further ionization action and replenishing the lost number of ions.

Low-temperature non-equilibrium plasma is characterized by a relatively low degree of ionization, a parameter showing the ratio of the concentration of ions to the total concentration of particles in the gas phase. Although the ionization degree can vary widely depending on the discharge conditions, an approximate value of 1 charged particle per million neutral is often taken as a rough estimate. Accordingly, only about one-millionth of the total number of particles in the gas phase is available to participate in sputtering, a small number at reduced pressure. Even though one ion can knock out more than one atom from the surface depending on the cathode material and ion energy, the total number of sputtered atoms is also small and the rate of metal film formation is low. Thus, diode sputtering has not been accepted as a practical tool for thin film deposition.

The situation changed in the 1970s when magnetron sputtering was invented. It is based on the idea that if it is difficult, under the given conditions, to increase the energy of the bombarding ions to sputter more atoms, it is necessary to increase the probability of the ionization events near the cathode (also called “the target” in the sputtering community). The goal was achieved by adding a ring of permanent magnets behind the cathode, which created a specifically configured, ring-shaped non-homogeneous magnetic field above the target surface. The secondary electrons emitted from the surface move along complex spiral trajectories due to the Lorentz force given by the local superposition of the electric and magnetic fields. Their mutual configuration forces the electrons to be confined within the ring tunnel, drifting and hopping inside. Hence, the electron travel time in the vicinity of the target substantially increases, and so does the ionization probability. Each ionization event produces a positive ion and the electron, and, thus, the plasma density becomes higher. Consequently, the higher fluxes of ions bombard the target surface, yielding much higher fluxes of sputtered atoms and increasing their deposition rate.

How can one use the peculiarities of thin film growth to nano-engineering of the surface? The following two extreme models should be recalled here:

• a layer-by-layer (Frank–van der Merwe);

• island (Volmer–Weber) growth.

Both models compare the interaction energy between an ideally flat initial surface and incoming atoms adsorbing on this surface (adatoms). If the adatom–surface interaction energy is higher than that of adatom–adatom interactions, adatoms tend to spread over the surface, forming the first layer. The second layer starts growing after the first layer has been completed; thus, the layer-by-layer growth occurs, leading to an atomically flat surface. Although this regime is crucial in many technologies, especially in the epitaxial growth of semiconductor materials, we are more interested in nano-structured materials in this Tutorial. The Volmer–Weber model describes how adatoms interact stronger with each other than with the surface atoms, forming islands on the surface. Subsequent growth of these islands results in the formation of nano-structured thin films with a rough growing front. Thus, one of the prerequisites for nano-structuring is the dominance of cohesion (like–like adatom interaction) over adhesion (adatom–surface interaction).

The above models describe the growth under equilibrium conditions, considering only the energetical aspects (the difference in the material properties) and not the kinetic aspects (the deposition rate and the surface diffusion rate). Many existing deposition processes, including magnetron sputtering, occur far from equilibrium, meaning that an adatom does not have sufficient time to find an energetically favorable site on the surface before it encounters another adatom. Therefore, non-equilibrium deposition processes offer the additional physical parameters to tune the surface morphology: the flux of adatoms (controlled by the deposition rate) and the adatom surface diffusion (controlled by the substrate temperature).

Empiric analysis of the non-equilibrium thin film growth resulted in the creation of the so-called zone structural diagrams. Movchan and Demchishin were the first to suggest the use of a parameter T/T_m, where T is the substrate temperature and T_m is the melting temperature of the deposited material, to describe the typical morphologies observed in thin metal films prepared by vacuum evaporation [Fig. 8(a)].⁸⁴ It turns out that most of the metals form nano-structured coatings at T/T_m < 0.3; i.e., if the substrate temperature is sufficiently low to hinder the surface diffusion and favor the island formation. The initial islands grow into domed columnar structures during the further deposition, widening toward the top and leaving a substantial unoccupied space in between [zone 1 in Fig. 8(a)]. The void fraction reaches 30% in this case. At higher substrate temperature, the films become more dense and compact, and structural zones 2 and 3 can be distinguished. Their realization is critically important for numerous applications of continuous thin films but will be omitted in this Tutorial, focusing on nano-structures.

Markedly, magnetron-sputtered metals form thin films with morphologies that can also be generalized into structural zone diagrams, similar to that of Movchan and Demchishin. Thornton constructed such a diagram, introducing an additional parameter of the Ar pressure used during the sputtering [Fig. 8(b)].⁸⁵ The Ar pressure determines the mean free path of species in the gas phase and their collision frequency. Thus, hyperthermal metal atoms sputtered from the target lose their energy in collisions with neutral Ar atoms to a greater or lesser degree, depending on the pressure. At a lower Ar pressure, metal atoms arrive onto the substrate with excessive energy and dissipate it, facilitating the surface atom re-arrangement. At a higher Ar pressure, metal atoms thermalize in collisions with neutrals and arrive on to the substrate with reduced energy, thus attenuating the surface diffusion and facilitating the island formation. Hence, it is the region of lower substrate temperature and higher Ar pressure [zone 1 in Fig. 8(b)] that is effective for the columnar growth and nano-structuring of the surface.

Later, Anders modified Thornton's diagram to account that energetic incoming species may contribute to significant redistribution of material on the substrate, including re-sputtering [Fig. 8(c)].⁸⁶ The idea stemmed from the analysis of highly energetic depositions, such as High-Power Impulse Magnetron Sputtering (HiPIMS), at which substrates are often subject to intensive bombardment by energetic species, including ions. In this diagram, the temperature ratio is approximated by a generalized temperature T*, which also comprises the potential energy of arriving species. The Ar pressure is replaced with a normalized energy E* that describes the kinetic energy of arriving species. In addition, the third axis is introduced, which refers to the net film thickness t*. A remarkable outcome of the Anders diagram is that the film thickness may go to negative values if the energy of bombarding species is too high, reflecting the onset of ion etching. Concerning this Tutorial's topic, however, the most attractive region is a zone where the surface diffusion is limited, the energy of the incoming species is low, the resultant film thickness is high, and a large void fraction characterizes the film structure.

The structuring of thin films can be enhanced further if the deposition is performed under an oblique angle (also called glancing angle deposition, GLAD). In this case, embryonic islands formed on the surface during the first moments serve as obstacles to the incoming atomic flux, accepting impinging atoms and creating shadow areas behind. Thus, well-separated columns grow on the surface [Figs. 9(a) and 9(b)]. Collimated atomic fluxes and low substrate temperatures are preferable for better column separation. The best results were obtained with ultrahigh-vacuum (UHV) evaporation, at which the mean free path is high, scattering on residual gases is low, and parallel atomic fluxes can be obtained by using Knudsen cells and collimators. If the position of the substrate is changed in a zig-zag manner or the substrate is rotated during GLAD, it is possible to achieve a diverse morphology of the resultant structures. Spirals, helices, zig-zag, and many other structures were reported [Figs. 9(c) and 9(d)].^87,88 The deposits are said to be nano-sculptured, highlighting the researchers’ ability to shape the growing objects as if by precise sculpting.^89,90

Magnetron sputtering can also be adapted to GLAD; however, the method requires higher pressure to maintain the discharge, and the resultant structures are typically worse defined as compared to UHV approaches. Nevertheless, vast examples exist now, demonstrating that careful tuning of the magnetron sputtering conditions can lead to the formation of nanorods (nanocolumns) of various materials. Starting with metals, GLAD was extended to metal carbides, oxides, and nitrides using reactive magnetron sputtering [Figs. 9(e)–9(h)].^91–102 The approach appears very attractive for the applications in which a highly developed surface is required, such as photovoltaics, photoelectrochemical water splitting, catalysis, and many others.

GLAD structures may also prove feasible in terms of bactericidal activity. For example, Ti nano-spikes can be created by sputter-GLAD on the surface of clinically used implants [Figs. 10(a)–10(c)],¹⁰³ and such nano-spikes can damage adhering bacteria, acting especially effective against Gram-negative E. coli and not much effective against Gram-positive S. aureus [Figs. 10(d)–10(f)].^103–106 The resultant structure mimicked the nano-pillar structure of Clanger cicada wings (Psaltoda claripennis) that was recently shown to kill bacteria solely by rupturing the bacterial cell membrane and not by chemical means.^72,73,107 The flat Ti surface does not exhibit bactericidal activity, and therefore the bactericidal action of the GLAD Ti was also attributed to such a biomimetic physical mechanism. At the same time, the flat and GLAD Ti surfaces were cell-compatible, showing good adherence, spreading, and the osteogenic response of mesenchymal stem cells [Figs. 10(g)–10(i)].¹⁰³ The combination of bactericidal activity and cell compatibility is crucial in developing advanced bone implants with reduced risk of inflammation and improved osseointegration.

The bactericidal effect of the GLAD structures can be substantially enhanced if Ti is replaced with metal, which is detrimental to bacteria per se. For example, silver-based nanocolumns induce several log reductions of E. coli concentration just in several hours [Figs. 11(a)–11(c)].¹⁰⁸ 

So far, bottom-up approaches to surface nano-structuring have been presented, in which atomic fluxes arrive onto the substrate and participate in self-organizational phenomena, leading to the formation of various nano-objects. Interfacial processes are crucial in this case. As an alternative, top-down methods can be suggested that produce nano-objects in a separate process and then deposit them onto substrates as ready-made entities. Gas-phase aggregation of nanoclusters and nanoparticles (NPs) has become one of the most attractive top-down approaches in the last few decades.

Although the specific mechanisms of the formation and growth of nanoparticles are still the subject of scientific discussion, the general rule is known: the material must be delivered to the gas phase, and super-saturation conditions must be created that trigger spontaneous condensation of the material's vapors. The well-known analogy was well illustrated by the example of the formation of clouds of altocumulus lenticularis over mountains.¹⁰⁹ The warm and humid air rises the mountain, cooling with altitude. Water vapors condense into liquid droplets, forming a cloud as the temperature goes below the dew point at a given pressure (super-saturation conditions). If the air masses go over the mountain and begin to descend, the temperature increases, and the droplets evaporate. Therefore, the cloud is seen to hang around or stick to the peak of the mountain. The same phenomenology can be used for the formation of metal NPs. Because the equilibrium vapor pressure of metals is extremely low under ambient conditions, it is necessary to employ high-vacuum or ultrahigh-vacuum systems and heat metals to at least the melting temperature or higher. Historically, evaporative systems were the first to be studied for the formation of metal NPs in the gas phase (the process is also called gas-phase aggregation). An alternative route involves magnetron sputtering of metal targets, which was first realized in 1991 by Haberland's group.^110,111 In this case, heating is avoided, and atomic metal vapors are delivered to the gas phase through the ion bombardment of the target surface. Argon is often used as an inert gas for sputtering; however, its pressure is typically higher than that in thin-film deposition to limit the out-diffusion of metal atoms from the aggregation zone, facilitating their thermalization. Sputtering is performed in a separate gas aggregation chamber divided from the deposition chamber by a system of orifices with or without differential pumping. Also, the walls of the aggregation chamber are cooled by tap water or liquid N₂ to achieve better super-saturation and facilitate the nucleation processes. The entire assembly of the vacuum chamber, the magnetron, and the exit orifice is called a gas aggregation cluster source (GAS, Fig. 12). The pressure difference forces the NPs to move with the gas flow from the GAS to the deposition chamber, where they are collected on substrates. A particular case is associated with the deposition of NPs onto the surface of vacuum-compatible liquids such as polyethylene glycol. As a result, solvent-, residual-, and linker-free nanofluids can be produced, in which only two components are present: metal NPs and host liquid.¹¹² 

A great variety of single-metal NPs were produced by this approach.^114–126 The recent years witnessed the efforts to synthesize bi- or multi-metal NPs of various morphologies. The experimental arrangement becomes more complicated in this case. It involves the sputtering of a composite target consisting of two (or more) metals,^127–132 multi-magnetron sputtering with several magnetrons installed in a single aggregation chamber,^{113,133–138} or in-flight coating of pre-formed NPs by shells of a counter-metal using an auxiliary magnetron in a separate modification chamber (Fig. 12).^139–143 Depending on the experimental conditions and metal miscibility, various NP shapes featuring different phase separation phenomena can be realized. For example, Cu-Ni nanoalloys were synthesized by letting a beam of Ni NPs pass through a hollow tubular magnetron with a Cu target.¹⁴² The phase diagram shows good miscibility of these two metals, and indeed homogeneously mixed Cu-Ni NPs can be produced, even though Cu atoms arrive onto the surface of already-formed Ni NPs [Figs. 13(a) and 13(b)].¹⁴² By contrast, the binary mixture of Cu and Ag demonstrates a large miscibility gap in the phase diagram. A similar deposition of Cu on pre-formed Ag NPs in the same setup results in the formation of Janus-type NPs, in which both metals are phase-separated into equal halves [Figs. 13(c) and 13(d)].¹⁴³ It was also shown that the morphology of binary NPs with a substantial lattice mismatch could be arrested at a dumbbell heterostructure by choosing the proper thermal conditions in the GAS. For example, body-centered cubic Fe segregates from face-centered Ag with the formation of the dumbbell-shaped NPs [Figs. 13(e) and 13(f)].^144,145 Under different conditions, core–shell or Janus-type Fe-Ag NPs are possible. Recently, a remarkable example of multi-framed cubic Fe-Au NPs was demonstrated [Figs. 13(g) and 13(h)].¹³⁸ The approach used simultaneous sputtering from two independent magnetrons with Fe and Au targets. The resultant multi-frame morphology was given by a complex interplay between the independent nucleation of Fe and Au NPs, their coagulation, and site-specific wetting of Fe facets with Au atoms. As a consequence, cubic Fe NPs are formed, which comprise well-defined and separated thin layers of Au.

Sputter-based GASs can be adapted for the fabrication of hybrid NPs that combine metals with polymers or semiconductors. Figure 14(a) shows the TiO₂-paraffin core–shell NPs. TiO₂ NPs were produced by reactive magnetron sputtering of Ti in Ar/O₂ mixtures using GAS, whereas paraffin nanoshells were deposited in-flight by thermal evaporation using an auxiliary evaporation chamber.¹⁴⁶ Reactive magnetron sputtering can also be implemented by introducing an organic precursor directly into the gas aggregation chamber. In the case of Ag target sputtered in the mixture of Ar, O₂, and hexamethyldisiloxane, the concurring processes of the formation of Ag NPs and PECVD of the organosilicon plasma polymer result in the growth of multicore–shell hybrid NPs [Fig. 14(b)].¹⁴⁷ Furthermore, rf magnetron sputtering of polymers was used to produce plasma polymer NPs, which can be in-flight decorated with metal NPs using auxiliary dc magnetron sputtering. Otherwise, a composite polymer-metal target can be employed. Figures 14(c) and 14(d) show satellite-type NPs prepared in this way consisting of a nylon-sputtered polymer core decorated with many smaller Ag or Cu NPs.^148,149

All-inorganic core–shell NPs can be produced by sputtering transition metals, by merely exposing the resultant metal NPs to ambient atmosphere and allowing an oxide shell to form over the metal core as shown in Fig. 14(e) for V-V₂O₅ NPs.¹³⁵ Metal oxide NPs can also be produced by reactive magnetron sputtering in Ar/O₂ mixtures.¹⁵⁰ As an alternative, reactive magnetron sputtering of transition metals can be performed in Ar with N₂. For example, Ta₃N_yO_x NPs can be produced in this manner, with the chemical composition and crystallinity of the NPs controlled by the gas-phase composition [Fig. 14(f)].¹⁵¹ If two immiscible metals are co-sputtered using two independent magnetrons, the formation of core–shell NPs is possible, as shown above, and here in Fig. 14(g). For two metals with different reactivity, such as Au and Ti, the Ti shell can be deliberately oxidized by introducing O₂ to the deposition chamber. Thus, intact Au NPs become enveloped by TiO₂ semiconductor shells.¹³⁶ Finally, inorganic compound NPs can be produced by sputtering composite targets consisting of two elemental precursors. Magnetron sputtering of Mn and Si targets was shown to produce Mn₅Si₃ NPs;¹⁵² the homogeneous mixing of the two types of atoms in a single compound NP is shown in Fig. 14(h).

With regard to anti-bacterial applications of gas-phase aggregated NPs, the research has focused so far on well-known Ag and Cu NPs and their embedding into bearing polymer matrices for tunable release of Ag⁺ and Cu²⁺ ions. The particular examples will be presented in Sec. III B 5.

The choice of the method of anti-bacterial surface engineering strongly depends on the demands of the mechanisms of coatings action, as it was discussed in Sec. II, but also often dictated by technological demands. In that regard, two distinguishable plasma technological approaches are often considered: (i) low-pressure plasma processing and (ii) atmospheric pressure plasma methods. Both approaches are very different not only in the way how plasma is generated and applied for surface modification but also have several fundamentally different pathways of plasma/surface interactions. In general, low-pressure plasma is capable of precise control over the ion energy, types of species, and species flux toward the treated surface. Therefore, low-pressure plasma is often used as a source of ions and reactive species with high energy applicable for surface etching or surface sputtering. Whereas, in a contrast, in high pressure plasma the energy of charged particles is transferred very effectively to the rest of the gas mixture because of a very high frequency of collisions. This results in rather low ions energy achieved in atmospheric pressure discharge and such plasma is often considered a “chemical reactor” in a gas phase that can provide a high concentration of active species with rather low energy for either surface etching or plasma polymerization. In Sec. III B, the focus will be given to the use of the plasma polymerization approach in the development of anti-bacterial coatings. Here, we will only discuss the use of atmospheric pressure plasma for nano-patterning through the methods of chemical etching. It has to emphasize that the use of plasma at high pressure for the development of well-defined 3D surfaces is still at its beginning stage and the amount of works, as well as our understanding of the processes initiated by such discharges, is less elaborated in comparing with low-pressure plasma methods discussed early.

Dimitrakellis et al.¹⁵³ presented the concept of the combined synthesis of organic–inorganic nanocomposites and atmospheric pressure plasma etching. The approach was used for the manipulation of surface topography and the fabrication of multifunctional surfaces such as superhydrophobic surfaces. In the work, atmospheric plasma etching in a dielectric barrier discharge (DBD) device operating in He/O₂ gas mixture was applied to achieve surface patterning based on the generation of reactive species under plasma action. The authors were also able to incorporate ZnO particles in the surface that can provide a strong anti-bacterial effect because of the presence of Zn ions. The general schematic of the method applied is illustrated in Fig. 15.

A similar approach based on the use of reactive oxygen species generated in DBD discharge in He/O₂ mixture has been applied in another work.¹⁵⁴ As a first step, atmospheric pressure plasma was used to selectively etch organic material (i.e., cellulose fibers) and enhance the micro- and nano-scale topography. It was found that the high etching rates of organic matter deriving from the atmospheric pressure plasma conditions resulted in the removal of the top cellulose fibers in a very short treatment time (3–4 min). Obtained materials have a surface roughness helping to engineer superhydrophobic surfaces that, in principle, can be used to avoid bacteria attachment to the materials.

The use of He/O₂ plasma for nano-patterning was demonstrated and mechanisms of etching were analyzed in Ref. 155. The authors made a compilation of their results dedicated to the etching of different polymeric materials under atmospheric pressure plasma conditions. They successfully applied plasma etching in order to manipulate the size of colloidal particles coated on silicon surfaces, aiming to produce etching masks for nano-patterning applications. An example of obtained patterns is presented in Fig. 16.

In Ref. 156, a combination of atmospheric and low-pressure etching has been used to prepare nano-patterning of SiO₂ surfaces for the purposes of the semiconductor industry. The same approach can also be applied to engineer anti-bacterial materials with desirable surface morphology but the costs and feasibility of the method still have to be assessed. The first steps in this direction have been made by the same team¹⁵⁷ revealing the bactericidal effect of micro-/nano-textured surfaces against Gram-negative bacteria (E. coli).

Despite some progress in the development of nano-texture materials by the use of chemical etching under atmospheric pressure plasma conditions, the methods still need a more systematic study directed to both understanding the mechanism behind the plasma-surface interactions and biological activity of the surfaces prepared. In Sec. III B, other methods to prepare anti-bacterial surfaces by functionalization and plasma polymerization are described and analyzed.

Plasma-based approaches can not only modulate the surface topographies with nano-structures as presented above but also provide a versatile platform for regulating the surface chemistry that can be used for fabricating anti-bacterial products. In this section, the typical antifouling coatings, drug-release coatings, and quaternary ammonium coatings prepared by plasma-based approaches are illustrated. In particular, the emerging aerosol-assisted plasma deposition (AAPD) method is highlighted.

Generally, cold plasma processing allows treating of the thermo-sensitive biomaterials due to its mild processing conditions that can not only remain the already optimized bulk properties (e.g., mechanical properties) but also improve the response properties of a material to its surrounding environment (e.g., antifouling property). Since the treated materials are exposed to a highly reactive plasma environment which comprises excited atomic, molecular, ionic, and free-radical species, several processes such as plasma activation, plasma polymerization, and plasma-induced graft polymerization (Fig. 17) can be determined depending on the use of plasma types, chemical monomers (also called precursors), operation conditions, etc.¹⁵⁸ 

Briefly, plasma activation, Fig. 17(a), commonly uses a gas discharge plasma for direct treatment of a material to introduce desired functional groups so that the surface properties such as surface wettability and surface adhesion can be changed expectedly. Typically, most kinds of gases such as N₂, O₂, or NH₃ are widely employed to introduce oxygen- or nitrogen-containing functional groups, which can usually result in surface hydrophilization. Inert gases, argon or helium, are also commonly used in plasma activation that can create free radicals on polymer surfaces and incorporate functional groups when exposed to ambient environment subsequently.¹⁵⁹ Some free radicals created by plasma activation provide covalent bonds that can initiate a grafting process with a suitable monomer/precursor under certain conditions, leading to forming plasma polymer.¹⁶⁰ The two-step process combined plasma activation with other polymerization techniques (e.g., traditional wet chemistry) is known as plasma-induced graft polymerization (sometimes also called plasma grafting/plasma graft) [see Fig. 17(c)]. In contrast, plasma polymerization [Fig. 17(b)] is another famous plasma-based technique to fabricate a thin coating for biomedical applications. When appropriate precursors are injected into a discharge plasma area in either a liquid or vapor phase, precursor fragments, reaction, and recombination can occur in plasma, which can result in depositing a plasma polymer coating. The first studies on organic vapor plasmas date back to 1874 when the Thenards published an earliest known report on the condensation of an organic solid from hydrocarbon vapors upon the action of what we know now as dielectric barrier discharge.¹⁶¹ 

It is not necessary to inject the precursor to a discharge plasma area directly, although a one-step process can be enabled in this way for most cases. The precursors can be absorbed on a surface first with some methods (e.g., immersion or spraying), and the surface is treated subsequently by plasma for polymerization. Anyway, the coating properties can be determined by the precursor nature and plasma conditions. The operation seems to be easy, but the coating formation mechanism remains a challenge for different kinds of precursors, which is still under investigation in the past decade.^162,163

It has to be emphasized that there can be some connections between plasma activation and plasma polymerization. For instance, Ar + O₂ plasma activation as pre-treatment of a polymer surface can efficiently improve the cytocompatibility of plasma-polymerized acrylic acid coatings;¹⁶⁴ Ar plasma activation used as post-treatment of the plasma hexamethyldisiloxane (HMDSO) deposition can incorporate oxygen-containing functional groups to render hydrophobic plasma coating with hydrophilic properties.¹⁶⁵ 

Another type of bioactive nano-engineered surface can be prepared by using NPs polymerized in plasma. The occurrence of organic NPs in plasmas was first documented in the mid-20th century,^166–168 and they were extensively investigated under the agenda of dusty plasmas; however, the benefits of NPs as building blocks for nano-engineered surfaces were acknowledged not so long ago.

In plasma polymerization, the synthesis of NPs proceeds via the recombination of radical and/or ionic carbonaceous species. These are formed as a result of electron impact-driven bond cleavage in volatile organic molecules. Under sufficiently high pressure, the mean free path is small enough to ensure a high collision frequency between the gaseous species and favor radical recombination in the gas phase. Newly born species may undergo further cycles of plasma activation and recombination and that is how plasma polymer nuclei and then NPs are formed.

Plasma polymer NPs eventually deposit on surfaces adjacent to the plasma, including electrodes, and such deposits used to be often considered an unwanted shortcoming. However, the configuration of GAS can be adapted to synthesize such NPs in a separate vacuum chamber, with the gas flow preventing the sedimentation of NPs on the walls and providing their continuous extraction to the deposition chamber where they can be deliberately collected on substrates.¹⁶⁹ NPs can be produced in GAS either by plasma polymerization of volatile organic precursors^170–174 or by rf magnetron sputtering of polymer targets.^175–177 For example, nylon-sputtered NPs were already shown in Figs. 14(c) and 14(d) as nano-beads decorated with Ag or Cu NPs.

As discussed in Sec. II, antifouling surfaces are one of the valid strategies to prevent bacterial attachments on a surface. Plasma-induced graft polymerization can be very helpful to prepare such antifouling coatings for preventing bacteria attachment and inhibiting biofilm formation. For example, a typical non-fouling PEG coating was successfully fabricated via the plasma-induced graft polymerization process on both polyamide (PA) and polyester (PET) surfaces through silicon tetrachloride (SiCl₄) plasma activation in low-pressure and subsequent liquid-phase PEG immersion.¹⁷⁸ The plasma activation step is very critical due to the high reactivity of SiCl₄-origin plasma species that can provide appropriate anchors for the subsequent grafting process. The results in this study showed that a 96% reduction in L. monocytogenes biofilm formation was observed on both PEG-grafted samples compared with their corresponding unmodified control. Similarly, different molecular weights of PEG can also be grafted on a surface with excellent antifouling performance using a similar plasma grafting approach.¹⁷⁹ 

Plasma-induced graft polymerization may also be very versatile that can be used for multifunctional surface fabrication. As an example, the implantable silicone rubber surface was activated by Ar plasma activation first and submerged in a chemical synthesized allyloxy PEG₂₄₀₀-polyhexanide (APEG₂₄₀₀-PHMB, molecular weights of the APEG = 2400) oligomer aqueous solution under a UV light (see Fig. 18).¹⁸⁰ The grafted APEG₂₄₀₀-PHMB bottlebrush-like coatings showed dual antimicrobial (contact-killing) and antifouling functionalities against Gram-negative/positive bacteria and fungi and also showed biocompatibility toward mammalian cells.

On the other hand, plasma polymerization could be much easier for synthesizing antifouling coatings due to the high feasibility of the simple injection of a precursor into the plasma area directly. As illustrated in Fig. 19, the monomer of 2-methyl-2-oxazoline (MOXA) was injected in a vapor phase into an atmospheric pressure He plasma jet system through a bubbling system also using He as carrier gas.¹⁸¹ The plasma-polymerized coatings on silicon substrates with different temperatures (50, 80, and 120 °C) were tested in this study for antifouling purposes. Consequently, an 80% reduction in the adhesion of S. epidermidis was achieved at the center of the plasma coating for the 120°C samples. Likewise, different chemical precursors such as hydroxyethyl methacrylate (HEMA), polyethylene glycol methacrylate (PEGMA), ethylenediamine (EDA), and ethylene glycol (EG) have also been fabricated using the plasma polymerization approach for fabricating antifouling surfaces.^47,48,182

Two or more bubbling systems are also possible to be used in one plasma system for achieving composite coatings with different chemical precursors in a one-step process. This is generally quite difficult for traditional wet chemistry approaches. For example, two kinds of precursors (PEG and EDA) were carried by two Ar bubbling systems separately to feed them together into a plasma reactor (Fig. 20).¹⁸³ The precursors were heated using a temperature control system for better vaporization. By varying the mixing ratio of the PEG and EDA precursors, the copolymers can be controlled over the amine density with excellent preservation of the internal PEG structure, leading to a dramatic reduction of non-specific protein adsorption. The combination of different types of precursors for fabricating composite coatings in a one-step process could be interesting and efficient for antifouling surface engineering, which represents a future research direction.

In addition to the one-step plasma polymerization process, it is also possible to use the two-step process that combines plasma activation with plasma polymerization to fabricate a better antifouling coating on biomedical devices. For example, Ti-based dental implants were coated with non-fouling PEG-like coatings through a two-step plasma process.¹⁸⁴ In the first step, Ar plasma activation was performed to enhance Ti surface hydrophilicity and surface adhesion in a low-pressure plasma reactor. Then, Ar was used to carry the tetraglyme monomer into the same plasma chamber through a bubbling system for plasma polymerization. The formed PEG-like coatings on the Ti surface showed both anti-adhesion properties for S. sanguinis and L. salivarius bacteria and biocompatibility toward fibroblasts and osteoblasts cells. Therefore, this study confirms that plasma-based approaches are eminently advisable for conferring Ti-based biomaterials with highly suitable properties for dental applications.

PEG-like coatings can also be prepared by Plasma-Assisted Vapor Phase Deposition, in which conventional poly(ethylene oxide) (PEO, similar to PEG) is heated under rarefied Ar.^{45,46,185,186} Thermal decomposition leads to a release of oligoethers into the gas phase where they can be additionally activated by low-temperature plasma. Depending on the discharge power/evaporation rate ratio, thin films with tunable cross-link density and, as a consequence, antifouling properties can be prepared.

Such films can be loaded with metal NPs, as discussed later in Sec. III B 5, or plasma polymer NPs to tailor the immobilization of bioactive molecules. For example, acrylic acid was plasma polymerized with the formation of NPs enriched with the carboxyl groups (ppAA NPs).^172,173 The ppAA NPs were fixed on the substrate surface by overcoating them with a thin film of PEO plasma polymer (ppPEO), Fig. 21(a).¹⁷⁴ Apart from anchoring the NPs, ppPEO fulfills the role of a non-fouling material, resisting the accumulation of biomolecules. When put into an aqueous solution, the ppAA/ppPEO nanocomposite absorbs water, which leads to the dissociation of the COOH groups on the surface of ppAA NPs. If the ppPEO overcoat is thin enough (about 3 nm), the negative charge from the carboxyl ions manages to propagate through the capping layer and attract positively charged biomolecules, provided that such are present in the solution. The phenomenon is demonstrated in Fig. 21(b), showing the liquid-cell AFM images of positively charged lysozyme molecules accumulated locally at the sites where the ppAA NPs reside. It should be noted that ppPEO withstands the immobilization of lysozymes in the regions between the NPs, providing spatially localized biomolecule attachment. The amount of the adsorbed lysozyme is proportional to the number of deposited ppAA NPs, Fig. 21(c). It is expected that the electrostatically attached lysozyme can be released reversibly back to the solution upon the change in pH below the isoelectric point. It is also worth mentioning that the uncovered ppAA NPs escaped from the surface of ppPEO when immersed in the solution. This phenomenon can be used to deliver drugs to cells. This application was demonstrated in the case of plasma polymerization in C₂H₂/N₂/Ar mixtures, Fig. 21(d).¹⁸⁷ The NPs were shown to contain unquenched free radicals that can be regarded as a benefit for the attachment of biomolecules without chemical linkers. Figure 21 shows that multiple functionalizations of NPs can be achieved with different immunoglobulins or other biomolecules, reportedly without deterioration in biological activity and molecular conformation. The functionalized NPs may be used as carriers of bioactive cargo to cells, penetrating the cell membranes and accumulating in the cytoplasm.

Until now, all the above examples of antifouling coatings are based on the hydrophilic properties of a surface. In fact, in addition to these hydrophilic coatings that enable antifouling functionalities due to a high affinity for water, plasma polymerization can also be useful to prepare superhydrophobic surfaces to inhibit bacteria, proteins, and/or microorganisms attachment on a biomedical surface.¹⁷ Typically, fluorinated and silicone coatings can impart a surface with superhydrophobic properties, and some of them can have antifouling functionalities. For instance, it has been reported that plasma-polymerized superhydrophobic 1H,1H,2H,2H-perfluorodecyl acrylate (PFDA) coatings exhibited protein repellent behavior against three model proteins; i.e., ovalbumin (OVA), human serum albumin (HSA), and fibrinogen (FGN).¹⁸⁸ The biomaterial, polyurethane (PU), has been modified with a pulsed-DC plasma polymerization method using HMDSO/ CF₄ mixture as the precursor and the fabricated superhydrophobic surfaces showed remarkable antifouling properties with no platelet adhesion and fibrinogen adsorption.¹⁸⁹ Moreover, Stallard et al. have investigated the protein adsorption and bacteria attachment on plasma-polymerized coatings with different surface wettability ranging from superhydrophilicity to superhydrophobicity that was achieved with different siloxane and fluorosiloxane precursors; i.e., tetramethylethosilicate (TEOS), HMDSO, and the mixture of tetramethylcyclotetrasiloxane (TMCTS) and perfluorooctytriethoxysilane (PFOTES).¹⁹⁰ The superhydrophobic siloxane and fluorosiloxane surfaces showed minimal indication of protein adsorption and the fluorosiloxane superhydrophobic coatings on plain titanium surface showed almost no bacteria attachment even after protein adsorption (Fig. 22).

Nevertheless, we have to admit that the anti-bacterial properties of some superhydrophobic surfaces are short-lived and the rough nature of these superhydrophobic surfaces may lead to the failure for their long-term use.¹⁹¹ Therefore, the coating durability and long-term antifouling properties for this kind of anti-bacterial surface should be at the center of future research focuses.

In recent years, coatings capable to release anti-bacterial drugs have attracted much attention due to their efficient bactericidal features with a reduced possibility of causing bacteria antibiotic resistance. In this regard, plasma treatment can be very useful in tailoring material surface properties that can meet the high requirements of practical biomedical applications. The released chemical compounds from a coating generally include reactive oxygen and nitrogen species (RONS), antibiotics, and metal-based anti-bacterial agents which have been extensively investigated in the field of plasma materials processing.⁶² 

With the development of modern biology, the understanding of the roles of RONS in regulating various physiological functionalities of living organisms has been reinforced, particularly in antimicrobial functionalities.^192,193 Briefly, the RONS are generated in an innate immune response and are of crucial importance for host resistance to microbial pathogens. Materials with the capacity to release these biomimetic RONS can be very desirable for anti-bacterial applications. Among many types of RONS release materials, nitric oxide (NO) released biomaterials may be one of the most studied ones.^56,194 Actually, in addition to anti-bacterial functionalities, NO is also known as a “gasotransmitter” that plays a crucial role in many physiological events such as the signaling for pain perception, sleep control and regulation, and vasodilation.¹⁹⁵ Ideally, integrating appropriate NO molecules onto a biomaterial surface can avoid significant limitations in direct delivery (e.g., NO is easily inactivated by oxygen or hemoglobin due to its reactivity). Releasing the loaded NO molecules in a controllable manner can keep their functionalities until the interactions with the local target.^196,197

Plasma polymerization has been proposed to produce NO release coatings for anti-bacterial applications.¹⁹⁸ In this study, isopentyl nitrite was used as a vapor precursor in a pulsed plasma for polymerization. The pulsed plasma enables the reservation of enough functional nitrosoxy groups in the molecular activation and keeps the functional groups in a stable coating in the air. Once the coating is immersed into an aqueous media, these functional groups can react with water and release the appropriate NO gas. The results showed that the amount of released NO was dependent on the immersion media (pH) and temperature. The plasma polymer coating showed a bactericidal effect for inhabiting S. epidermidis biofilm formation for as long as 14 h and no cytotoxic side effect to human mesenchymal stem/stromal cells. These properties are extremely important advances as the functionalities of NO are dose-dependent and the concentration has to be adjusted to obtain the desired therapeutic effect.¹⁹⁹ 

Probably for most cases, plasma-polymerized coatings are more frequently used as the targeted surface anchoring sites for loading functional molecules for specific applications. Amine-based plasma coatings have been reported to corporate diazeniumdiolate (NONOate) into the coatings.²⁰⁰ In this example, two kinds of commonly used amine-based precursors (i.e., allylamine or diallylamine monomers) were used for plasma polymerization to provide the mainly primary or secondary amine functionalities on the treated surfaces. The formed amine plasma coatings were then exposed to NO gas under pressure. This can facilitate the primary and secondary amine groups on the base coating reacting with NO molecules to form NONOates as the NO donor [Fig. 23(a)].²⁰⁰ The coatings were very stable in the air and only showed their active properties when in contact with aqueous media. Both kinds of plasma coatings continuously released the NO for over 48 h under different temperatures in phosphate-buffered saline (PBS, pH = 7.4) solution [Fig. 23(b)] and showed significant inhibition effects for both Gram-negative P. aeruginosa and Gram-positive S. aureus [Figs. 23(c) and 23(d)]. This study also emphasized that the loading and release amount of NO can be tailored by adjusting the thickness of the plasma coating so that the best anti-bacterial effects combined with the lowest cytotoxicity could be achieved.²⁰⁰ This statement was confirmed by a subsequent study.²⁰¹ In the study,²⁰¹ the plasma-polymerized 3-mercapto-3-methylbutan-1-ol with thiol functionalities was converted to S-nitrosothiol and the coatings with different thicknesses were capable of releasing a different amount of NO molecules in a controlled way for 48 h.

A plasma polymer can also be used to store and release antibiotics for anti-bacterial to a local infection site with a high dose so that the efficiency can be enhanced and side effects can be avoided when compared with systemic clinical treatments. Generally, the antibiotics are loaded onto a biomedical material surface followed by a plasma polymer, which acts as a diffusion barrier that can not only prevent elution of the loaded drugs by solvents and excludes contamination but also control the drug-release kinetics.^202,203 A typical example is shown in Fig. 24.²⁰⁴ First, a biocompatible porous anodic alumina oxide (AAO) was used for loading Vancomycin. Then, the top layer was covered by the plasma allylamine polymer coating. The loaded drugs can be released in a solution environment and the release time can be tailored by changing the coating thickness (deposition time). The drug was completely released within 45 min for the uncoated sample while increasing the time of deposition had led to the slower release, and only half of the loaded drugs were released after 500 h for the coating with 200 s deposition time.²⁰⁴ This strategy thus provides an innovative strategy for effective controlled drug release over an extended period, which can ensure continuous migrating and prophylactic protections for the target sites in practical implantation applications. Analogously, a plasma-polymerized PEG-like coating was used to cover an ampicillin-loaded beta-tricalcium phosphate (β-TCP) bioceramic, and the release kinetics of the loaded drugs were changed ideally that can avoid burst release and slow down the initial rate of release with the maintained activity.²⁰⁵ 

Considering the easy control over surface chemistry by plasma polymerization using different deposition precursors, the plasma polymer barrier can also provide more functionalities to respond to an external environment that can enable smart release kinetics. For example, dual polymer layers composed of plasma-polymerized 1,7-octadiene (ppOct) and plasma-polymerized acrylic acid (ppAA) were capped on a biocompatible porous silicon drug reservoir that was first loaded with levofloxacin (LVX, it is a broad-spectrum antibiotic commonly used for wound infection treatment in the first line).²⁰⁶ The LVX release behavior of the plasma-fabricated devices is pH-dependent. When pH = 5, LXV release was inhibited, but it can be under control when pH > 8. Moreover, the released LVX from the device retained its antimicrobial activity against the P. aeruginosa wound pathogen with an 80% reduction in bacterial growth for up to 16 h, showing great potential in bacterial infection wound healing applications.

In some cases, plasma activation can also play a significant role in the drug loading process. For example, a He plasma jet at atmospheric pressure has been employed to treat a β-TCP bioceramic and a slow release profile of the loaded doxycycline hyclate was achieved due to the newly formed populated bonds and charges induced by the plasmas.²⁰⁷ Air plasma activation has led to an enhanced drug (ampicillin) loading capacity of a surgical polypropylene (PP) mesh due to the plasma-induced new surface chemistry and improved wettability.²⁰⁸ 

Instead of using the coating as a diffusion barrier of these ready-to-release chemical compounds (like antibiotics discussed above), the plasma-polymerized coating can also be used as a polymer matrix for embedding the functional anti-bacterial nanoparticles. In this regard, incorporating the metal and metal oxide NPs (Ag, Cu, ZnO, TiO₂, and many others) into a plasma coating is at the center of the relevant research.^202,209,210 Moreover, it is also possible to entrap antibiotics (e.g., by aerosol-assisted plasma deposition, see Sec. III B 3) for specific anti-bacterial applications.

So far, the exact mechanisms for these metal-based anti-bacterial agents are still not clear, but in general, it is due to their capability to release bactericidal metal ions and RONS.^211–214 Anyway, it is very useful to incorporate them into a polymer coating to impart biomaterials with anti-bacterial functionalities, and plasma surface engineering may play a significant role in the fabrication. Noticeably, among many anti-bacterial metal NPs, Ag NPs may be one of the most studied ones in the plasma surface engineering community, but almost all the fabrication protocols are similar that could be shifted from one to another.

Frequently, plasma deposited polymer films can be used for the covalent attachment of these metal anti-bacterial NPs but it generally requires multiple steps. In a recent example, Ag NPs were successfully covalently attached to a plasma-polymerized radical-rich thin film in a three-step process [Fig. 25(a)].²¹⁵ First, a mixture of acetylene, argon, and nitrogen gasses was used for plasma polymerization on a Ti surface in a slightly changed plasma polymerization setup which was under an enhanced bombardment of accelerated ions achieved through pulse biasing of the substrate. The change allowed an enhanced cross-linking degree, yielding high concentrations of radicals embedded within the coating structure. In the second step, the plasma polymer coated Ti surface was simply immersed into an AgNO₃ solution. After that, UV irradiation was adopted as the last step. In the study,²¹⁵ the method of plasma polymerization was also compared to other coating techniques; i.e., dip coating and electrophoretic deposition. The plasma-polymerized coating showed a low and sustained release of Ag⁺ over 14 days [Fig. 25(b)], which confirmed the firm covalent bonding between the nanoparticles and the plasma-polymerized layer that can mitigate the release of nanoparticles and excess free ions in the medium. Furthermore, the coating fabricated by plasma polymerization showed the highest anti-bacterial efficiency over 14 days [Fig. 25(c)] and the best adhesion of mammalian cells compared to the other two coating techniques [Figs. 25(d)–25(g)].

On the other hand, it can be much easier to prepare nanocomposite coatings capable of releasing metal ions by directly injecting the metal NPs together with organic precursors into a discharge plasma area. For example, Deng et al. prepared Ag NPs containing nanocomposite coating in a single step via directly feeding the Ag NPs together with tetramethyldisiloxane (TMDSO) into an atmospheric pressure plasma jet, which resulted in a high bactericidal effect for both E. coli and S. aureus strains.²¹⁶ However, with the development of precursor injection methods in recent years, the more frequently used plasma-based approach for enabling one-step synthesis of such metal-containing coating has become the recent emerging aerosol-assisted plasma deposition. Therefore, more detailed information on this emerging technique will be presented together with more examples of drug-release coatings in Sec. III B 3.

Aerosol is a suspension of liquid or solid particles in a gaseous medium, with droplets size below 100 μm, and more typically below tens of micrometers.²¹⁷ 

Aerosol generators, also known as atomizers or nebulizers, can be easily and advantageously coupled to plasma sources for plasma polymerization. In plasma polymerization, the main concern is the selection and injection of suitable chemical precursors, when handling solid and, in general, low vapor pressure chemicals. Aerosol-assisted plasma deposition (AAPD) overcomes such issues, since with this method each precursor sufficiently stable and compatible with reactor materials, can be used. In fact, liquid or solid precursors can be fed to the plasma, pure or dissolved in a solvent via atomization. Further advantages of plasma polymerization in the presence of aerosol are a higher deposition rate, due to the high mass transport rate of the precursor, and multi-precursor injection with good stoichiometric control.

Hence, AAPD can be really advantageous for depositing bioactive coatings, such as antimicrobics, from antibiotic molecules or nanoparticles that cannot be easily vaporized in the plasma.^{132,218–221}

We can distinguish three different approaches to supply an aerosol feed to a plasma source, whatever the configuration is, as illustrated in Fig. 26.²²¹ In approach I, a monomer or its solution (Ia) or a dispersion of an active compound (Ib, e.g., a drug) is injected in the form of an aerosol in the plasma. In approach II, the aerosol simply supplies an active additive (e.g., solid nanoparticles or a drug) but the precursor for the plasma polymerization is added through an auxiliary line.

Whatever the approach, chosen AAPD is very versatile:

1. Monomer vapor pressure is not a problem;

2. Additive and monomer can be chosen independently, hence several combinations can be found leading to different nanocomposite coatings;

3. The additive can be soluble in the aerosol liquid feed, but it can be organic or inorganic (metals or metal oxides) nanoparticles.

In this case, the liquid will be likely a suspension and continuous stirring during aerosol generation is necessary to keep a constant composition of the driven drops. From a practical point of view, it should be considered that in method Ib, two different building blocks are fed with one atomizer, but their relative abundance can only be changed by using atomizer liquid of different compositions. On the other hand, using two different atomizers to supply the two components of a composite coating, changing the relative flow rate will more easily result in a different composition of the resulting film.^222–224 

In some modified AAPD setups, the aerosol is injected remotely, outside the electrode zone. Therefore, as reported in Fig. 27,²²⁵ droplets impinge on the substrate, without passing through the plasma region. In this case, the sample is typically placed on a movable stage, and it is moved alternatively from an aerosol injection section to a plasma one. When the sample is in contact with the aerosol, the precursor is adsorbed onto the sample surface, but then moved to the plasma source, where the adsorbed layer reacts with the impinging active species (such as excited neutrals and photons).

AAPD can be very useful for preparing non-fouling polysaccharide or PEG-like coatings since the usual precursors are quite non-volatile.^226–228 In fact, for the latter case, typically monomers are tri- or tetra-glymes, or similar compounds, bearing the –CH₂CH₂O– repeating unit, with a boiling point in the range 150–200 °C, more easily injected in the plasma by atomization. In a comparative study, tetraglyme was remotely injected via aerosol (AAPD) or with a bubbling system (APPE-CVD), in atmospheric pressure argon plasma, to prepare protein-resistant coatings.²²⁶ For coatings deposited by APPE-CVD, a decreased retention of the PEG functionality was observed when the power input was increased. On the other hand, the retention of the monomer structure in AAPD was higher and almost independent from the power input (in the investigated range). Also, the corresponding protein adsorption resistance was higher when the monomer was atomized. This is due to the higher mass transport rate leading to a lower energy/molecule ratio, resulting in a lower dissociation of the precursor.

As an alternative to the preparation of anti-bacterial coatings, AAPD can be used to deposit film bearing functional groups that can bind active molecules or nanoparticles. As an example, the deposition of cathecol-containing films from an aerosol of N-(3,4-dihydroxyphenyl)acrylamide (DOA) dissolved in vinyltrimethoxysilane (VTMOS) can lead to the deposition of a functional coating that can bind Ag NPs or antifouling/anti-bacterial molecules like dispersine or ranaspumin-2.^229,230

More easily, anti-bacterial NPs can be deposited in the form of nanocomposite coatings by feeding the aerosol with a suspension of such NPs and the addition of a monomer.²³¹ In the example illustrated in Fig. 28,²³¹ ethanol dispersed Ag NPs were sprayed in Ar plasma jet, and acrylic acid was added with a bubbling system. In this way, the monomer (acrylic acid) polymerized and the simultaneously impinging Ag NPs were entrapped in the growing film, forming a nanocomposite. By testing such coatings against E. coli, anti-bacterial activity was assessed. An analogous coating can be obtained even by replacing Ag NP with its salt water solution (AgNO₃). Recently, Wang et al.²³² prepared fabrics coated with films containing silver nanoparticles by atomization of a AgNO₃ solution and addition of an organosilicon monomer.

These are a few examples, but similar metal-containing composite coatings can be easily prepared from salt solutions or NP particles, with any combination of metal and matrix precursor.

Another class of composite coatings, that can be efficiently deposited by AAPD, is that of antibiotics containing films. Lysozyme, a natural antiseptic enzyme, vancomycin and gentamicin, two common anti-bacterial drugs, have been plasma embedded in a hydrocarbon matrix.^233–236 In this case, the anti-bacterial compound is injected into a DBD system in the form of an aerosol, from their water solution. Ethylene, but other precursors can be considered as well, forming the matrix embedding the drug, which is released when in a humid site, developing the anti-bacterial activity. Interestingly, these systems can form a nanocapsule-shaped coating as illustrated in Fig. 29.²²³ 

Details of the formation mechanism of such unique (in the field of plasma deposition processing) nano-features can be found elsewhere.²³⁷ Briefly, this can be ascribed to a balance between solvent evaporation and solute molecules diffusion in the aerosol droplets, leading to segregation of the solute to the drop surface and deposition of a thin plasma polymer (where ethylene is the precursor in this case) around them. Some further examples of AAPD will be illustrated in Sec. III B 4.

Quaternary ammonium compounds (QACs) are known as antimicrobics since the 1930s.²³⁸ QACs at that time were the main first-line defense against pathogenic microorganisms. The main moiety in the chemical structure of QACs is represented by quaternary nitrogen (N⁺), since the N atom is covalently bonded to four groups, giving, hence, the molecule an ionic character. Commercially available compounds that can be found in consumer goods are benzalkonium chloride and cetyl pyridinium chloride, used as a skin and oral disinfectant. QACs are effective against both Gram-positive and Gram-negative bacteria, acting as contact-killing coatings. The exact antimicrobial mechanism of QACs has not been fully understood. However, the predominant mode of action is through disruption of the cell membrane due to the strong ionic character of the compound. Important is the molecular weight of QACs and, in particular, the length of the alkyl chain linked to the quaternary nitrogen, making the compound act as an ionic surfactant able to penetrate the cell membrane. A similar mechanism works for the disruption of membrane-equipped viruses. In the case of pathogenic fungi, having a more complex and resistant cell membrane, the same mechanism could not stand, and the antimicrobic activity can be related to the cationic charge inhibiting a good adhesion of the fungi, a step necessary to their proliferation.²³⁸ 

Direct plasma polymerization of a QAC-like coating is rarely reported, though it can be achieved quite easily by aerosol-assisted plasma deposition. More often alternative options are proposed. A possibility is that of depositing an amino-rich coating and then converting N atoms in quaternary ammonium groups by a wet reaction. Jampala et al. deposited onto stainless steel a plasma coating from a gas mixture containing hexamethyldisiloxane and ethylene diamine.²³⁹ After the reaction in hexyl bromide solution, the quaternary ammonium groups were formed, and the coating was active in reducing the growth of K. pneumoniae.

More often plasma is used for “activating” the polymer surface: the reaction with non-polymerizable gas, such as Ar, air, or O₂, breaks surface bonds with the formation of radicals and polar groups such as OH. Such surfaces can then be used to enhance the adsorption/grafting of QACs as illustrated in Fig. 30(a).

Air atmospheric pressure plasma has been used to treat PE foils and PET/cellulose wipes,^240,241 to induce linkage of QAC. In the case of PE foils, the plasma-treated samples were immersed in a vinyl quaternary ammonium solution salt, to induce graft polymerization. In this case, the 300 nm polymerized film was stable and provided excellent activity against a selection of Gram-positive and Gram-negative bacteria according to the ASTM E2149-13a standard test method.²⁴⁰ Song et al.,²⁴¹ instead, upon air plasma treatment, confirmed improved adsorption of alkyldimethylbenzylammonium chloride of PET-rich wipes. This resulted in contact anti-bacterial behavior against Gram-positive S. aureus and Gram-negative E. coli, ATCC 25923 bacteria.

Another approach can be followed for immobilizing QAC on a surface. The substrate can be immersed in a solution of the disinfectant, and after drying, exposed to a plasma fed with air, oxygen, or argon. During the latter step, cross-linking of the QAC is induced, and grafting to the substrate as well. This is particularly effective if double bonds (e.g., vinyl groups) are present in the structure of the QAC. This sort of plasma “curing” method is illustrated in Fig. 30(b), in the case of methyl diallyl ammonium (MDAA) salt.²⁴² In that work they compared samples obtained from plasma-induced graft polymerization with those produced by immersion and following plasma curing, using similar conditions. They found that the latter was faster in producing samples with a better anti-bacterial effect.

To produce more stable QAC coatings by this plasma curing method, a cross-linker can be added to the QAC compound, such as a molecule bearing at least two vinyl groups. Malshe et al. applied this process onto Nylon-Cotton fabrics, impregnating them with diallyldimethylammonium chloride, and used pentaerythritoltetraacrylate (with four vinyl groups) as a cross-linker.²⁴³ After an air atmospheric pressure plasma jet treatment, they obtained coated fabrics showing a 99.9% reduction in the bacterial colonies of K. pneumoniae and S. aureus.

However, the most direct and practical way to coat a material with a QAC coating is to use aerosol-assisted plasma deposition, described in Sec. III B 3. In fact, to inject in the plasma a solution of quaternary ammonium salt with an atomizer is straightforward. Salts of cetalkonium chloride, benzalkonium chloride, and cetylpyridinium chloride were dissolved at 2.5% w/w in acrylic acid or poly(ethylene glycol methacrylate) and poly(ethylene glycol) diacrylate, and atomized in pilot-scale atmospheric pressure plasma apparatus, as illustrated in Fig. 31, in order to coat fabric substrates. The coating resisted washing at pH 2, 7, and 12, and microbial tests demonstrated that cetylpyridinium chloride and benzalkonium chloride-containing coatings provided significant protection against fungal contamination.²¹⁸ 

Plasma-polymerized coatings can also be useful in building multi-layers, in some cases with an all-plasma processing method. One can think to build a sandwich system in which anti-bacterial nanoparticles are trapped between two coatings having a regulatory role: the first layer acting as an adhesive interface with a substrate and the top layer having the role of controlling the release rate of the anti-bacterial agent. Plasma processing offers the possibility to have three consecutive reactors each producing the bottom, the active, and the top layer respectively, exactly an all-plasma method. Just to give an example the bottom and top layers can be organic plasma deposited coatings; sandwiching Ag nanoclusters deposited by plasma sputtering. However, more frequently the solution proposed for the active layer is a wet step, where the substrate coated with the bottom layer is placed in contact with a solution or suspension of anti-bacterial NPs or antibiotics.

Vasilev et al. proposed to sandwich Ag NPs between two layers of plasma-polymerized n-heptylamine (HApp), the bottom one 100 nm thick while the overlayer was less than 20 nm, as depicted in Fig. 32.²⁴⁴ The Ag NPs were grafted onto the bottom HApp layer by dipping in a AgNO₃ solution and following reduction to metal state with NaBH₄. They demonstrated control over the silver release, by modulating the thickness of the overlayer (Fig. 32) with such a multi-layer approach, and they proved complete inhibition of S. epidermidis. Furthermore, they showed that the coatings were compatible with osteoblast cells, which is important in medical device applications.

A similar multi-layer system has also been proposed by Alissawi et al.²⁴⁵ They deposited a bottom Teflon like coating by a low-pressure plasma sputtering source, and Ag nanoparticles were deposited by thermal evaporation. To control the release of the active particles, a coating was plasma-polymerized from HMDSO/O₂/Ar feed. They observed that the addition of the top layer can slow down the release rate in the water of silver ions. Much more, by changing the oxygen/HMDSO flow rate ratio, they could tune the release, because the coating passed from hydrophobic (no O₂) to more hydrophilic (20 SCCM of O₂). In the latter case, it can be supposed that water can interact with the top coating leading to the easier release of the silver.

An interesting approach to multi-layer has been proposed by Kulaga et al.²⁴⁶ They embedded silver NP from AgNO₃ solution (and following reduction in NaBH₄) between two organic layers onto a flexible substrate. The bottom one was obtained from low-pressure plasma polymerization of maleic anhydride and immersion in water for conversion in –COOH-rich coating. Then, the silver loaded sample was coated with a similar coating. The authors demonstrated that combining this coating with electron beam sterilization can lead to the formation of microcracks in the multi-layer (without delamination) that helps in regulating the release of the anti-bacterial agent.

Another approach uses GAS described earlier in Sec. III A 3 to load metal NPs into plasma polymer matrices. NPs can be deposited sequentially or simultaneously with the matrix; consequently, multi-layer sandwich structure or homogeneously mixed nanocomposite thin films can be produced. The controlled ion release is achieved by tuning the number of embedded NPs and the thickness of the polymer capping layers. For example, bi-layered Cu NPs and thin films of PEO plasma polymers were produced [Figs. 33(a) and 33(b)], which showed the bactericidal activity not only against common bacteria but also against multi-drug-resistant bacteria, that is, those that survive antibiotic administration, such as Methicillin-Resistant Staphylococcus aureus (S. aureus, MRSA) and Pseudomonas aeruginosa (P. aeruginosa), as shown in Fig. 33(c).²¹⁰ The presence of the polymer phase allows these coatings to be applied to flexible substrates, including wound dressing materials, and thus to cope with a serious issue of hospital-acquired infections for patients with open wounds and invasive devices.

Typically, a higher concentration of Cu or Ag NPs ensures a more decisive bactericidal action, as shown in the example of Cu NPs embedded in the fluorocarbon plasma polymer matrix, Fig. 33(d),²⁴⁷ and it would be instructive to aim for an increase in the NP load. However, one should keep in mind that biological tissue-contacting devices must meet the criterion of biocompatibility; that is, their performance should be harmless to cells. Such a demand exerts greater restraint on the ion release kinetics, and a balance should be found between the bactericidal activity and the cytocompatibility. For Cu NPs/C:F and Ag NPs/C:H [Figs. 33(e)–33(h)] nanocomposite coatings,^248,249 such a compromise has been found: the optimized films demonstrated distinct anti-bacterial properties against E. coli and good cytocompatibility with MG63 and MC3T3 cells.

This Tutorial aims to provide researchers with a comprehensive overview of plasma processing methods for engineering anti-bacterial surfaces. The focus of the Tutorial is made on backgrounds of plasma methods of surface treatment that are demonstrated with some typical examples of applications in the biomedical field. The examples given in the Tutorial have revealed plasma's unique potential as a versatile tool for very effective control of the surface chemistry and surface morphology—the two key factors that determine the application area of novel materials.

The Tutorial gives an overview of the plasma-enabled processes for both low-pressure (vacuum-based) and atmospheric pressure conditions and the entire spectrum of common materials from metal alloys and ceramics to polymers and bio-scaffolds. Impressive recent advances in plasma functionalization of surfaces suggest that plasma-assisted surface functionalization approaches are promising to produce anti-bacterial materials for targeted bactericidal applications ranging from medical tools and biomaterials to all kinds of surfaces used in everyday human life. The plasma-modified surfaces have revealed the ability to actively control the interactions with bacteria, e.g., by contact-killing, preventing the interaction through antifouling properties or controlled drug release.

With this Tutorial, the authors aim to give a detailed and systematic description of different plasma methods and also provide rationality for a choice of the plasma methods depending on both requirements of bacterial killing mechanisms and properties of materials important in real-life applications. The unique features of the plasma-assisted surface processing methods are emphasized in the Tutorial with a specific focus on anti-bacterial materials development. The key plasma features are discussed in the Tutorial and physical manipulation of surfaces on atomic or nano-scale (e.g., sputtering processes in magnetron discharge) or chemical manipulation with surface chemistry (e.g., by chemical etching or polymerization).

Importantly for practical applications, plasma technology of anti-bacterial surfaces engineering has several notable features. The methods of plasma processing are very fast, not based on the use of solvent, only affect the top surface with negligible impact on the bulk properties, effective in functionalization of atomic/nano-/micro-scale, easily scalable to industrial demands, and environment-friendly with potential applicability in the broad range of materials.

Nonetheless, in some cases, the plasma-engineered antimicrobial surfaces may suffer from some disadvantages. Frequently encountered problems include the poor coating adhesion and stability in an aqueous media that may disable the biomaterials and the extensive use of nanoparticles that may cause a long-lasting toxicity impact on cells. Therefore, comprehensive consideration of both advantages and disadvantages of plasma surface engineering should be at the center of designing nanocomposite and antimicrobial biomaterials with relevant plasma methods.

The fast development of new plasma sources and our understanding of the mechanisms of plasma-surface interactions give the researchers a diversity of possible solutions in the field of anti-bacterial materials design and to solve future challenges in the field. Among them, we can emphasize new trends in the development of a top-down approach where new plasma processing of anti-bacterial materials can be designed based on final requirements to the materials. Such an approach, different from the current paradigm of bottom-up experimental methodology, can be combined with plasma physics study and plasma-surface modeling and helps to save required time and costs for the development of new plasma methods of anti-bacterial materials engineering. Second, the multifunctional anti-bacterial materials capable to combine different mechanisms of bacteria growth suppression are in high demand and can help to design the next generation of anti-bacterial surfaces but more systematic research is required.

In summary, plasma engineering of surfaces has several unique features allowing to manufacture a variety of anti-bacterial materials for almost all kinds of applications. The plasma-based methods of bactericidal surfaces engineering provide cost-effective, environmentally friendly, and scalable solutions for both research and industry with potential still to be fully discovered.

A.N. acknowledges the Vlaio project HBC.2019.0157: In-line kwaliteitscontrole van plasma-gedeponeerd dunne films voor industriele toepassing. A.C. appreciates the support from the Czech Science Foundation via Grant No. GACR 21-12828S.

The authors have no conflicts to disclose.

The data that support the findings of this study are available within the article.