**2. Plasma generation and designed devices for skin disinfection**

CAPs could be generated in lab using plasma source and driven by power supplies with working gas. Working gases such as noble gases (He, Ar), N<sup>2</sup> , O2 , air, or their mixtures could be used to ignite the plasma. Typical constituents of plasma include electrons, ions, neutral particles (background gas molecules), UV radiation (UBA, UVB, UVC), heat, reactive oxygen, nitrogen species (RONS), and so on (see **Figure 2**). All these components within plasma make CAPs highly reactive. The constituents and concentration of species in plasma depends on the plasma source, the power input as well as the working gas. When contacting with microorganisms or cell/tissue, components of plasma will play different roles in the treating processes; however, the mechanism has not been fully understood yet. It is well known that UVB (280–320 nm) and UVC (100–280 nm) could be able to cross the epidermis, and UVA (320–400 nm) can even reach the dermis. UVB and UVA can also trigger skin cancer. However, a lot of reports claimed that UV radiation in plasma is low and does not play a significant role in anti-microorganism process except that from microwave-driven discharge [15–18].

**Figure 3** summarizes several typical plasma sources used in different research groups. (a) is called plasma pencil designed by Laroussi group [30]. This device driven by sub-microsecond high-voltage pulse uses He gas flowing through a modified DBD device (with holes in the center of dielectric plates), therefore the plasma created between electrode panels comes out and forms a plasma plume up to 5 cm. Another famous plasma source construct is a floating-electrode DBD device (FE-DBD) which uses the treating object as the second electrode and could generate plasma between the electrode surface and the substrate [31]. FE-DBD could directly use the air inside the short gap as working gas. Safety and stability of FE-DBD largely depends on the power supply, the gas gap, and the electrode shape. Kolb et al. introduced a DC-driven microhollow cathode discharge to generate plasma plume outside the tiny hole (c) [32]. It could use air or other gas as working gas. A 51 KΩ ballast resistor is connected to the circuit to restrain the current to 20 mA. The dimension of the plume changes with the gas flow rate as well as the gas temperature. Lu et al. reported a single-electrode plasma jet powered by nanosecond pulse DC [33]. Helium is used as working gas and the length of plasma plume could reach up to 4 cm. The

sion spectra (OES) (d). Another structure called plasma needle usually uses bare metal needle as electrode, see (e) and (f) [9, 34]. (g) is a portable DC-driven plasma needle array device called plasma flashlight [35]. This device can directly use a DC battery to power up and create plasma in the open air. (h) is a plasma brush of relatively large area [36]. (i) is also a classical design

+

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are all detected by optical emis-

gas temperature is about 300 K and species like O, OH, and N<sup>2</sup>

**Figure 3.** Typical CAP sources.

**Figure 2.** Plasma components.

Charged particles in plasma were concluded to play an essential role in bacterial inactivation by rupturing the outer layer of cell membrane [19, 20]. Mendis et al. reported the electrostatic force caused by charge accumulation on the cell membrane could overcome the tensile strength which leads to rupture [15]. However, other researchers claimed different viewpoints that the anti-bacterial effect of the charged particles is due to the chemical modification of the membrane surface [21]. CAPs are usually designed to operate in near room temperature (less than 40°C); therefore, there would not be substantial thermal effects on microbial cells [22]. The effect of heat can be ignored. The microorganism is under indirect plasma treatment which means that plasma does not directly contact with the target; the electric field was too weak to contribute to the inactivation process. In some cases, when plasma is in direct contact with the samples, the electric field could become high enough to take effect [23]. However, in most of the cases, there is a rare possibility to put the samples within 300 μm away from the electrode to cause damage by the electric field [24]. If the plasma device is using air or O2 containing mixture or operating in the open air, RONS such as O, O3 , NO, NO2 , OH, and H<sup>2</sup> O2 would present. These highly active species are believed to play an important role in all the plasma treatment processes and have already been reported by many researchers [18, 26–28]. The detailed discussion of the roles of reactive species can be found in [29].

**Figure 3** summarizes several typical plasma sources used in different research groups. (a) is called plasma pencil designed by Laroussi group [30]. This device driven by sub-microsecond high-voltage pulse uses He gas flowing through a modified DBD device (with holes in the center of dielectric plates), therefore the plasma created between electrode panels comes out and forms a plasma plume up to 5 cm. Another famous plasma source construct is a floating-electrode DBD device (FE-DBD) which uses the treating object as the second electrode and could generate plasma between the electrode surface and the substrate [31]. FE-DBD could directly use the air inside the short gap as working gas. Safety and stability of FE-DBD largely depends on the power supply, the gas gap, and the electrode shape. Kolb et al. introduced a DC-driven microhollow cathode discharge to generate plasma plume outside the tiny hole (c) [32]. It could use air or other gas as working gas. A 51 KΩ ballast resistor is connected to the circuit to restrain the current to 20 mA. The dimension of the plume changes with the gas flow rate as well as the gas temperature. Lu et al. reported a single-electrode plasma jet powered by nanosecond pulse DC [33]. Helium is used as working gas and the length of plasma plume could reach up to 4 cm. The gas temperature is about 300 K and species like O, OH, and N<sup>2</sup> + are all detected by optical emission spectra (OES) (d). Another structure called plasma needle usually uses bare metal needle as electrode, see (e) and (f) [9, 34]. (g) is a portable DC-driven plasma needle array device called plasma flashlight [35]. This device can directly use a DC battery to power up and create plasma in the open air. (h) is a plasma brush of relatively large area [36]. (i) is also a classical design

**Figure 3.** Typical CAP sources.


O2

, OH, and H<sup>2</sup>

, NO, NO2

Charged particles in plasma were concluded to play an essential role in bacterial inactivation by rupturing the outer layer of cell membrane [19, 20]. Mendis et al. reported the electrostatic force caused by charge accumulation on the cell membrane could overcome the tensile strength which leads to rupture [15]. However, other researchers claimed different viewpoints that the anti-bacterial effect of the charged particles is due to the chemical modification of the membrane surface [21]. CAPs are usually designed to operate in near room temperature (less than 40°C); therefore, there would not be substantial thermal effects on microbial cells [22]. The effect of heat can be ignored. The microorganism is under indirect plasma treatment which means that plasma does not directly contact with the target; the electric field was too weak to contribute to the inactivation process. In some cases, when plasma is in direct contact with the samples, the electric field could become high enough to take effect [23]. However, in most of the cases, there is a rare possibility to put the samples within 300 μm away from the electrode to cause damage by the electric field [24]. If the plasma device is using air or O2

would present. These highly active species are believed to play an important role in all the plasma treatment processes and have already been reported by many researchers [18, 26–28].

containing mixture or operating in the open air, RONS such as O, O3

**Figure 2.** Plasma components.

124 Plasma Medicine - Concepts and Clinical Applications

The detailed discussion of the roles of reactive species can be found in [29].

remains unclear. Possible mechanisms proposed by researchers are: (1) electroporation- and oxidation-induced cell wall/membrane dysfunction, which leads to leakage of cellular components; (2) intracellular oxidation and nitrification causing protein damage and gene expres-

Bacterial killing effect by CAPs has been investigated for more than 20 years. It is found that CAPs could effectively inactivate different type of bacteria, including gram-positive and gram-negative, anaerobic, aerobic, or facultative anaerobic bacteria [40]. The response of bacteria to CAPs is species-dependent and the Gram-positive bacteria is usually more susceptible to CAPs treatment because of the difference of cell-wall components, which indicates that the CAP-induced damage to the cell membrane and cell wall may be a key factor of antibacterial effect. The most common bacteria found in skin and wound infection are *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Bacillus cereus*, and so on, which have been proved to be effectively inactivated by CAPs [41, 42]. Unlike drugs, another advantage of CAPs is that plasma does not show any resistance after multi-treatment against bacteria. Maisch et al. reported significant decolonization of methicillin-resistant *Staphylococcus aureus* (MRSA) and *Escherichia coli* without cell damage of a

sion disorder; and (3) direct DNA damage such as causing double-strand breaking.

pig skin sample [43]. Alkawareek et al. also found complete inactivation of MRSA [44].

*Candida lipolytica* on agar surface using N2 + O2

face using a He + O<sup>2</sup>

investigated as well [49–51].

Many fungi are common constituents of skin flora, and under certain conditions, they would cause diseases. The effects of traditional tools such as chemicals, UV radiation, or heat are often unsatisfactory and sometimes accompanied by undesirable side effects. Unlike bacteria, fungus is more resistance to plasma treatment because of the much more complex cell biology. In 2008, Akishev et al. published the first paper of CAP decontamination of *Aspergillus niger* and

zone of 30–40 mm was observed [45]. Xiong et al. also inactivated *Candida albicans* on agar sur-

on the petri dish and found that restraining active species inside the chamber largely improved the antifungal effect. Daeschlein et al. used a low-temperature atmospheric pressure plasma jet to treat clinical isolates of *Trichophyton interdigitale*, *Trichophyton rubrum*, *Microsporum canis*, and *Candida albicans*. They found that plasma irradiation could eradicate fungal growth and no isolate exhibited resistance to plasma treatment [47]. In a new research area of plasma treatment of onychomycosis, Xiong et al. used three kinds of CAPs to treat *E. coli* and *Trichophyton rubrum* living on the back side of a nail model and found that bacteria is easier to inactivate than fungus and the inactivation effect also related to the structure of plasma sources [48].

Researchers also use CAPs successfully inactivate various virus and mechanism has been

It is known that more than 60% of all infections are caused by bacteria in the form of bacteria which could become resistant to treatment and often develop into a chronic state. A biofilm is often formed by a cluster of cells encapsulated by a 3D extracellular matrix (ECM), [52] which forms a good protection barrier for antibiotics and plasma agents. Therefore, cells inside this community have been demonstrated to exhibit higher antibiotic resistance than planktonic cells. However, CAPs have also shown great decontamination effect against biofilms with longer time than treating planktonic cells under same condition [53, 54]. Koban et al. compared the anti-biofilm (*Candida albicans*) effect by dielectric barrier discharge and plasma jet,

plasma jet [46]. They compared the antifungal effect of with/without a cap

plasma jet. After 30–60 treatments, inhibition

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**Figure 4.** Three commercialized CAPs medical devices.

(surface micro-discharge, SMD) representing 'indirect plasma' [37]. This type of plasma generates on the surface of the electrode, and the plasma does not directly contact with the treatment target. When using air as working gas, SMD could operate in three modes: call ozone mode, transition mode, and NOx mode, respectively. The transition between these modes depends on the input power. More detailed information of the different plasma sources can be found in [38, 39].

With the development of the lab-made plasma sources and fundamental studies, many commercial plasma device products have been delivered into the market. **Figure 4** shows three famous products for skin wound healing based on plasma jet device, FE-DBD and SMD, respectively. **Figure 4(a)** is the world famous kINPen plasma device, which is based on a RF argon plasma jet source and gained CE marketing certification in 2013. (b) is called PlasmaDerm based on FE-DBD. And (c) is MicroPlasma β, which origins from SMD device. The wound healing effect of these three products will be presented in the following sections.

### **3. Plasma interaction with microorganisms**

Plasma sterilization is the first research field of plasma medicine. Since Professor Laroussi published the first paper of plasma sterilization in 1996 on *IEEE Transactions on Plasma Science* [7], there came out thousands of studies on inactivation of bacteria, fungi, and virus using different plasma sources. At the same time, mechanism of plasma interaction with microorganism was studied by using physical, chemical, and biomedical diagnostic methods, such as optical emission spectra (OES), laser-induced fluorescence (LIF), Fourier transform infrared spectroscopy (FTIR), flow cytometry, electrophoresis, ELISA, chemiluminescence assay, and so on. However, the exact mechanism of plasma inactivation microorganisms still remains unclear. Possible mechanisms proposed by researchers are: (1) electroporation- and oxidation-induced cell wall/membrane dysfunction, which leads to leakage of cellular components; (2) intracellular oxidation and nitrification causing protein damage and gene expression disorder; and (3) direct DNA damage such as causing double-strand breaking.

Bacterial killing effect by CAPs has been investigated for more than 20 years. It is found that CAPs could effectively inactivate different type of bacteria, including gram-positive and gram-negative, anaerobic, aerobic, or facultative anaerobic bacteria [40]. The response of bacteria to CAPs is species-dependent and the Gram-positive bacteria is usually more susceptible to CAPs treatment because of the difference of cell-wall components, which indicates that the CAP-induced damage to the cell membrane and cell wall may be a key factor of antibacterial effect. The most common bacteria found in skin and wound infection are *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Bacillus cereus*, and so on, which have been proved to be effectively inactivated by CAPs [41, 42]. Unlike drugs, another advantage of CAPs is that plasma does not show any resistance after multi-treatment against bacteria. Maisch et al. reported significant decolonization of methicillin-resistant *Staphylococcus aureus* (MRSA) and *Escherichia coli* without cell damage of a pig skin sample [43]. Alkawareek et al. also found complete inactivation of MRSA [44].

Many fungi are common constituents of skin flora, and under certain conditions, they would cause diseases. The effects of traditional tools such as chemicals, UV radiation, or heat are often unsatisfactory and sometimes accompanied by undesirable side effects. Unlike bacteria, fungus is more resistance to plasma treatment because of the much more complex cell biology. In 2008, Akishev et al. published the first paper of CAP decontamination of *Aspergillus niger* and *Candida lipolytica* on agar surface using N2 + O2 plasma jet. After 30–60 treatments, inhibition zone of 30–40 mm was observed [45]. Xiong et al. also inactivated *Candida albicans* on agar surface using a He + O<sup>2</sup> plasma jet [46]. They compared the antifungal effect of with/without a cap on the petri dish and found that restraining active species inside the chamber largely improved the antifungal effect. Daeschlein et al. used a low-temperature atmospheric pressure plasma jet to treat clinical isolates of *Trichophyton interdigitale*, *Trichophyton rubrum*, *Microsporum canis*, and *Candida albicans*. They found that plasma irradiation could eradicate fungal growth and no isolate exhibited resistance to plasma treatment [47]. In a new research area of plasma treatment of onychomycosis, Xiong et al. used three kinds of CAPs to treat *E. coli* and *Trichophyton rubrum* living on the back side of a nail model and found that bacteria is easier to inactivate than fungus and the inactivation effect also related to the structure of plasma sources [48].

(surface micro-discharge, SMD) representing 'indirect plasma' [37]. This type of plasma generates on the surface of the electrode, and the plasma does not directly contact with the treatment target. When using air as working gas, SMD could operate in three modes: call ozone mode, transition mode, and NOx mode, respectively. The transition between these modes depends on the input power. More detailed information of the different plasma sources can be found in [38, 39]. With the development of the lab-made plasma sources and fundamental studies, many commercial plasma device products have been delivered into the market. **Figure 4** shows three famous products for skin wound healing based on plasma jet device, FE-DBD and SMD, respectively. **Figure 4(a)** is the world famous kINPen plasma device, which is based on a RF argon plasma jet source and gained CE marketing certification in 2013. (b) is called PlasmaDerm based on FE-DBD. And (c) is MicroPlasma β, which origins from SMD device. The wound healing effect of these three products will be presented in the following

Plasma sterilization is the first research field of plasma medicine. Since Professor Laroussi published the first paper of plasma sterilization in 1996 on *IEEE Transactions on Plasma Science* [7], there came out thousands of studies on inactivation of bacteria, fungi, and virus using different plasma sources. At the same time, mechanism of plasma interaction with microorganism was studied by using physical, chemical, and biomedical diagnostic methods, such as optical emission spectra (OES), laser-induced fluorescence (LIF), Fourier transform infrared spectroscopy (FTIR), flow cytometry, electrophoresis, ELISA, chemiluminescence assay, and so on. However, the exact mechanism of plasma inactivation microorganisms still

sections.

**3. Plasma interaction with microorganisms**

**Figure 4.** Three commercialized CAPs medical devices.

126 Plasma Medicine - Concepts and Clinical Applications

Researchers also use CAPs successfully inactivate various virus and mechanism has been investigated as well [49–51].

It is known that more than 60% of all infections are caused by bacteria in the form of bacteria which could become resistant to treatment and often develop into a chronic state. A biofilm is often formed by a cluster of cells encapsulated by a 3D extracellular matrix (ECM), [52] which forms a good protection barrier for antibiotics and plasma agents. Therefore, cells inside this community have been demonstrated to exhibit higher antibiotic resistance than planktonic cells. However, CAPs have also shown great decontamination effect against biofilms with longer time than treating planktonic cells under same condition [53, 54]. Koban et al. compared the anti-biofilm (*Candida albicans*) effect by dielectric barrier discharge and plasma jet, and used 0.1% chlorhexidine digluconate (CHX) and 0.6% sodium hypochlorite (NaOCl) as positive control. They found plasma treatment reduced the colony-forming units CFU significantly compared to chemical disinfectants [55]. In a later research, they investigated the synergistic effect of nonthermal plasma and disinfecting agents against single and multispecies dental biofilms. They found that the combination of plasma and agents increases the antimicrobial efficacy of all tested compounds [56]. Xiong et al. firstly used Laser Confocal Scanning Microscopy (LCSM) technology to obtain the depth of biofilm that plasma could penetrate through [57]. A He-O<sup>2</sup> mixture nanosecond pulse DC-driven plasma jet was used to treat a 10-day growth of *Porphyromonas gingivalis* biofilm, and found that 5-min plasma treatment could at least inactivate the bacteria under 15 μm. In their following work, they successfully inactivated a 25.5 μm biofilm using a plasma flashlight [35]. Puligundla and Mok reviewed the potential application of nonthermal plasmas against biofilm-associated microorganism in 2017. For more details about plasma interaction with biofilms, refer [58].

As mentioned earlier, the second stage of wound repair is related to cell proliferation and migration as well as angiogenesis. Cell types involved in wound healing are mainly fibroblasts and keratinocytes, among which keratinocytes contribute to the major healing processes and fibroblast cells play a guiding role [69, 70]. It has already been reported that CAPs could

simulated release of fibroblast growth factor-7 [71], as seen in **Figure 5**. Researchers from INP

**Figure 6.** (a) Non-invasive angiographic OCT images and (b) stereoscopic images longitudinally acquired over 14 days after wound generation; relative depth in angiographic OCT images is color-coded as from yellow (superficial) to red

(deep) [82].

/Ar microplasma through

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increases fibroblast cell proliferation and migration by using N<sup>2</sup>
