# Pulse Generator Characteristics Determine Helium/Oxygen Plasma Efficacy: An Insight Into Enhanced Bacterial Decontamination

Diego Morais da Silva, Augusto Stancampiano, Sébastien Dozias, Konstantin Georgiev Kostov, Cristiane Yumi Koga-Ito, Eric Robert

► To cite this version:

Diego Morais da Silva, Augusto Stancampiano, Sébastien Dozias, Konstantin Georgiev Kostov, Cristiane Yumi Koga-Ito, et al.. Pulse Generator Characteristics Determine Helium/Oxygen Plasma Efficacy: An Insight Into Enhanced Bacterial Decontamination. Plasma Processes and Polymers, 2025, 23 (1), pp.e70111. 10.1002/ppap.70111. hal-05495951

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# Pulse Generator Characteristics Determine Helium/Oxygen Plasma Efficacy: An Insight Into Enhanced Bacterial Decontamination

Diego Morais da Silva¹,² | Augusto Stancampiano² [ID] | Sébastien Dozias² | Konstantin Georgiev Kostov¹ [ID] |
Cristiane Yumi Koga-Ito¹ | Eric Robert²

¹São Paulo State University (UNESP), São Paulo, Brazil | ²GREMI/UMR 7344, CNRS/Université d'Orléans, Orléans, France

**Keywords:** antimicrobial activity | plasma jet | plasma medicine | reactive oxygen and nitrogen species | t-tube | tracheal stenosis

## ABSTRACT

This study examined two distinct pulse generators operating with the same reactor, utilizing helium and helium/oxygen mixtures as the working gases. The research focused on the distribution of RONS on the agar surface, their concentration, and the bacterial inhibition halo. The findings demonstrate that modifying the gas mixture influences the concentration of RONS, thereby altering the dominant reactive species and affecting the levels of others during plasma exposure. The incorporation of O₂ into the gas mixture leads to less homogeneous treatment, thereby complicating the eradication of microbes. Nevertheless, it also broadens the spectrum of RONS delivery, which may be beneficial for demanding clinical applications, including tracheal T-tube biofilm control, as preliminary investigated in this work.

## 1 | Introduction

Tracheal stenosis is a clinical condition characterized by a reduction in the diameter of the patient's trachea. If the normal trachea diameter is not recovered, many clinical complications can occur, such as respiratory insufficiency, infections, pneumonia, and, in extreme cases, asphyxia leading to death [1]. Tracheal stenosis stents, also known as T-tubes or Montgomery tubes, are used as an alternative treatment for tracheal stenosis [2]. The T-tube is inserted in this scenario because it serves as a stent to restore the normal tracheal diameter, enabling vital breathing [3]. Recently, cases of tracheal stenosis have been increasing post-COVID-19 pandemic, due to prolonged airway ventilatory support. In this context, the cases of post-intubation tracheal stenosis have been growing [4-7].

The T-tubes have many properties relevant to this application, like temperature and water resistance, biocompatibility,

malleability, and chemical inertness [2]. These properties are attributed to the medical-grade silicone used in the construction of the prosthesis. Otherwise, the biofilm formation susceptibility is one of the main limitations that compromise the T-tube performance [8]. The polymicrobial biofilm formed on the prosthesis is composed of fungi and bacteria due to contact with the tracheal mucosa and inhaled air [1, 9].

The formation of biofilm can lead to many clinical complications for patients, as local tissue damage increases the severity of the stenosis and hinders the recovery [5, 10]. Another complication is T-tube degradation due to microbial colonization. The biofilm degrades the prosthesis material, which usually requires replacement between 6 and 12 months after implantation. As the replacement requires a surgical procedure, it negatively impacts the patient's quality of life, apart from the generation of higher costs [1, 3].
Cold atmospheric plasma (CAP), especially in the jet configuration, has been studied as a promising technology in many medical applications [11–14]. The reactive oxygen and nitrogen species (RONS) and the UV radiation generated by the plasma demonstrated effectiveness in microbial inactivation. The antimicrobial activity of CAP was observed for different microorganisms, including *Escherichia coli*, *Staphylococcus aureus*, *Candida albicans*, and *Pseudomonas aeruginosa* [15–17]. Trebulová et al. used a plasma gun with a wire fed through a thin capillary to expose dog-ear models to CAP, demonstrating antimycotic activity [18]. CAP also showed low toxicity in exposure to various mammalian cell lines, which allows its safe clinical application [19, 20].

CAP has been studied as an alternative for biofilm decontamination [21–25]. The antimicrobial activity of CAP results from a combination of factors, such as the electric field, charged particles, UV radiation, and reactive species [26]. Xu et al. studied the application of CAP against *Staphylococcus aureus*, operating with 0.5% of O₂ added to helium gas, in a 10-min treatment. The CAP treatment resulted in a 5-log reduction in CFU/mL counting [27]. Baz et al. also observed CAP effectiveness against different pathogens (*Candida albicans*, *S. aureus*, *P. aeruginosa*, and *Candida auris*), in mono and multispecies models. The authors observed that CAP was effective against the biofilms in both in vitro models. The results indicated that the CAP effectiveness against *S. aureus* depended on the strain [28]. Udachin et al. studied the usage of an air CAP device for the disinfection of stainless steel in *S. aureus* and *E. coli* biofilm models. The CAP was applied for a maximum of 5 min and resulted in a maximum reduction of 4.3 log.CFU/mL for *E. coli* and 3.3 log.CFU/mL for *S. aureus* [29].

A previous study conducted by our research group examined the antimicrobial activity of CAP on mono- and multispecies biofilms grown on medical-grade silicone specimens [30]. The CAP treatment lasted 5 min at a working distance (distance from the plasma device to the sample) of 5 mm. The treatment demonstrated statistical significance for all the monospecific biofilms treated (*S. aureus*, *P. aeruginosa*, and *C. albicans*). The multispecies biofilm with the same strains exhibited a significant reduction only for *C. albicans* and *S. aureus*. The cytotoxicity of the protocol was also assessed for BEAS-2B cells (normal epithelial cells, isolated from human bronchial epithelium), and the protocol was found to be non-cytotoxic.

The present study aims to optimize the antibacterial effect and RONS distribution within the T tubes by varying the helium-to-oxygen ratio and the number of plasma pulses. These two parameters were not previously investigated in [30], due to some limitations of the plasma device. In this work, a new plasma jet configuration enables the critical assessment of these parameters for T-tube biofilm decontamination. Oxygen admixtures are known to enhance the antimicrobial activity of plasma, while pulse delivery control enables the reduction of plasma treatment duration. The new investigations and data documented in this work represent a step forward toward the clinical implementation of cold plasma technology for treating tracheal stenosis.

# 2 | Materials and Methods

## 2.1 | Plasma Devices Description

In this work, two cold atmospheric pressure plasma devices are investigated. The devices share the same jet reactor architecture but

are powered by two different high-voltage generators. Following the presentation of the typical plasma reactor, the two power supplies are described in the sections below. The purpose of comparing the two devices, BrD and NwD, is to provide a comprehensive understanding of the factors that affect the effectiveness of cold plasma in bacterial decontamination. This comparison is pertinent since both pulse generators exhibit different waveforms and levels of customization, with BrD being the only device available commercially, and it possesses fewer customization options than NwD.

### 2.1.1 | Plasma Jet Reactor

The device's principle of operation, its typical operational parameters, and schematic are reported in [17]. The plasma jet utilized for the experiments is produced within a conventional Dielectric Barrier Discharge (DBD) reactor featuring a metallic connector and a dielectric (glass) capillary, which is purged with helium (see Figure 1). The capillary exit is connected to a 1.0-m-long, 4-mm-diameter, flexible plastic tube equipped with an inner thin copper wire (1 mm in diameter) at floating potential. When the plasma in the DBD reactor is ignited, the Cu wire in contact with the plasma is polarized, and a slight secondary discharge forms at the other end of the Cu wire, a few mm before the plastic tube outlet. Consequently, a plasma plume propagates into the ambient air. This plasma jet, generated at the end of the long, flexible plastic tube, can be easily inserted into a cavity or small opening, making it particularly relevant to biomedical applications. This specific feature makes the device suitable for plasma application inside the T-tube implant. The reactor was powered by two different power supplies, as described in the following sections.

### 2.1.2 | Power Supply BrD

The plasma jet device described previously was driven by a commercial, low-frequency, high-voltage generator (Ibramed, Amparo, Brazil), initially designed for dermatological treatments. It produces a burst of several dumped sine waveforms (Figure 2A) with a maximal amplitude of approximately 12.5 kVp-p, which is repeated with the line frequency (50 Hz). The device features a switch that increments the number of sine waves in a burst from 1 to 5, effectively increasing the in-burst pulse repetition frequency to 250 Hz while maintaining the burst frequency (one to five individual pulses) at 50 Hz. This is the only way to modify the voltage signal parameters, which restricts the source's possible applications. On the other hand, this device is low-cost, easy to operate, and can deliver up to a couple of watts of power, making it suitable for biological applications.

### 2.1.3 | Power Supply NwD

The NwD power supply is a low-power (10 W) electrical generator that can deliver a controllable number of voltage pulses with a peak amplitude of 1–20 kV (10 kV used in this work) and a pulse duration of 4 µs. During our experiments, the device was operated at pulse repetition frequencies of 10 and 20 kHz (Figure 2C). Bursts of 10–100 consecutive pulses were produced at 50 Hz (20 ms period), corresponding to adjustable duty cycles between 5% and 50%. In this context, the “time-on” mentioned in Table 1 refers to the duration when the pulses are generated during a single modulation period. Conversely, the “time-off” indicates the duration when the pulses are not generated within
<figure><img src="image_2.png" /><figcaption>FIGURE 1 | Scheme of the plasma jet reactor employed during the experiments.</figcaption></figure>

the same modulation period. Therefore, each modulation period consists of consecutive pulses followed by a period of voltage time off (Figure 2B). The device is controlled by a computer, which allows selecting all the above-mentioned parameters, offering higher precision and customizability. Unlike BrD, NwD can deliver positive polarity pulses with a precise, constant peak amplitude for all pulses. Table 1 presents the parameters used during the pulse test using the NwD. The baseline protocol was selected to match the parameters provided by the BrD power supply (pulse frequency: 10 kHz; 10 pulses; burst frequency: 50 Hz). In the 10x protocol, the 20 ms was kept, but the number of pulses increased tenfold. Then, the 20x protocol represents an increasement in the factor of two (in the pulse repetition frequency) compared to the 10x setting, with the same duty cycle, to investigate if this increasement can allow a decrease in the treatment duration. For all the protocols, the voltage was set to 10 kV.

## 2.2 | Electrical Measurements

The high-voltage signals generated by both devices were monitored on a Tektronix Series 3 oscilloscope via a 1000:1 high-voltage probe (Tektronix P6015, bandwidth 75 MHz). The voltage temporal profiles for each configuration were obtained using Origin 2022 (OriginLab).

## 2.3 | Assessment of Reactive Oxygen Species (ROS) Production Using KI-Starch Agar

To obtain potassium iodide (KI) starch agar, a solution containing 2.2% agar, 0.3% KI, and 0.5% starch was prepared with demineralized water. The solution was autoclaved for 15 min, then 20 mL was distributed in each petri dish (ø 8.3 cm). After the agar solidification, the Petri dishes were stored at 4°C overnight until the plasma exposure experiment. The KI-starch sample acts as a broad-range oxidation reporter, sensitive to ROS (e.g., O₂, OH, H₂O₂, and O₃), but not selective or quantitative. The KI-starch method offers advantages, such as quick

setup and compatibility with liquid or solid substrates (including suspended and plated bacterial cultures and plasma exposure). When in contact with reactive oxygen species (ROS) from plasma delivery, KI-starch agar changes its color from yellow to blue due to I₂ formation, according to the following reaction:

$$2\text{ROS} + 2\text{KI} \rightarrow 2\text{K. ROS} + \text{I}_2 \quad (1)$$

$$\text{I}_2 + (\text{C}_6\text{H}_{10}\text{O}_5)_n \rightarrow \text{I}^*_2(\text{C}_6\text{H}_{10}\text{O}_5)_n \quad (2)$$

## 2.4 | Assessment of Reactive Oxygen Species (ROS) Formation Inside the T-Tube Using KI-Starch Gel

The formation of a thin layer of KI-starch gel inside the T-tube is a crucial innovation in this work. It represents a powerful tool for determining the spatial distribution and assessing ROS generation within the T-tube during and after plasma ignition. It provides qualitative information about the distribution of reactive oxygen species within the tube, thus allowing prediction of plasma action against the biofilm. Various tests were performed to deposit a stable, uniform agar layer on the inner wall of the tube. The final protocol for the gel deposit in the T-tube differs from that used in the previous agar experiments. A solution containing 2.2% gelatin was prepared, supplemented with 0.3% KI and 0.5% starch. The solution was then heated in a microwave oven until boiling. After 5 min of cooling, 1 mL of the solution was poured into the T-tube, which was connected to a rotative system. The system spins at low angular velocity (23 rotations per minute) until the agar is completely solidified. The agar was replaced with gelatin because it adheres more readily to the internal walls of the T-tube.

## 2.5 | Measurement of Ozone, Nitrogen Monoxide, and Nitrogen Dioxide Concentrations

The concentrations of O₃, NO, and NO₂ produced by each plasma-generation parameter used during the treatment were
<figure><img src="image_3.png" /><figcaption>**FIGURE 2** | The waveforms of: (A) BrD, several bursts (left), and the waveform of a single pulse (right). (B) Visualization of the signal electrical parameter (number of pulses, voltage time off, pulse repetition period, and signal modulation period). (C) NwD generator, operating with the Base, 10x, and 20x parameters, as an example.</figcaption></figure>
<table><caption>TABLE 1 | Parameters used to operate the NwD power supply.</caption><thead><tr><th>Protocol</th><th>Total number of pulses per second</th><th>Pulse repetition frequency (kHz)</th><th>Number of pulses in a burst</th><th>Time-off (ms)</th><th>Time-on (ms)</th><th>Duty cycle (%)</th></tr></thead><tbody><tr><td>Base</td><td>500</td><td>10</td><td>10</td><td>19</td><td>1</td><td>5%</td></tr><tr><td>10x</td><td>5000</td><td>10</td><td>100</td><td>10</td><td>10</td><td>50%</td></tr><tr><td>20x</td><td>10000</td><td>20</td><td>100</td><td>5</td><td>5</td><td>50%</td></tr></tbody></table>

measured. The ozone and NOₓ monitors APOA-360 and APNA-360 (Horiba) were used for each species, respectively. The gas inlet of the monitors was positioned 0.5 cm from the outlet of the plasma tube. The species were monitored for 5 min, the maximum treatment time, with the device continuously sucking gas through a flexible tube to the absorption cell, which quantified the concentration. The data was acquired every 12 s. The measurements were performed in triplicate.

## 2.6 | Temperature Measurements

During the procedure, a temperature probe (Optic Fiber OP-TOCON® FOTEMP1-4) was employed to measure the temperature in the region between the target and the plasma. The device features a measuring range from -200°C to +300°C with an accuracy of ±0.2°C. The sensitive tip of the probe is 1 mm in diameter and 1 cm long. The probe was positioned between the plasma and the agar at 5 mm, typically situated approximately 1 mm above the agar surface. The plasma exhibited a mean width of 1.3 mm. The temperature data were collected every 30 s for 5 min (the maximum treatment time). The gas temperature at the flexible tube outlet was monitored using both plasma devices with fixed He gas flow. By varying the number of HV pulses during NwD device operation, one can determine whether and how this parameter influences the gas temperature near the target sample.

# 3 | Results and Discussion

## 3.1 | Electrical Measurement

The voltage waveforms acquired from both devices are presented in Figure 2. The BrD signal exhibits two irregular voltage spikes repeated with a frequency of 50 Hz. A pattern can be observed: the second spike is always higher than the first, with amplitudes of about 10 and 7.5 kV (the delay between the two spikes), respectively. When examining each spike in detail, it can be confirmed that each constitutes a damped sine waveform with a period of approximately 100 microseconds. This corresponds to a pulse repetition frequency of 10 kHz, as illustrated in Figure 2C. The same pulse repetition frequency was adopted as the base parameter for NwD (10 kHz), with a pulse count of 10. This methodology was employed to assess the performance of both devices, which shared similar parameters in subsequent tests. NwD generator delivers an electrical signal composed of regular consecutive positive pulses, whose repetition frequency can be increased up to 20 kHz without any significant decrease in the pulse's peak amplitude. Additionally, the number of pulses can be adjusted from 10 to 100 (before reaching the time off), without affecting the peak amplitude, which remains

approximately 10 kV in all cases (Figure 2C). In this work, the device was operated with a pulse period of 0.1 or 0.05 ms, generating voltage bursts of 10 or 100 positive-polarity pulses. At these operating conditions, the power supply delivers stable pulses with a peak amplitude of approximately 10 kV throughout the entire 5-min treatment.

## 3.2 | Assessment of Reactive Oxygen Species (ROS) Using KI-Starch Agar

The KI-starch test was first performed to investigate how plasma jet operating conditions affect the formation and delivery of oxygen-reactive species generated by both devices during plasma treatment on the agar surface (0.5 cm from the tube outlet). The parameters studied were the treatment time (only for the BrD), gas composition (for both devices), and the number of pulses delivered (only for the NwD, as this parameter cannot be finely adjusted with the BrD).

As demonstrated in Figure 3, the region exposed to RONS expanded with increasing oxygen concentration for both devices, considering the NwD operating under the base parameters (as detailed in Table 1: pulse repetition frequency of 10 kHz, 10 pulses, 19 ms time-off, 1 s time-on, resulting in a duty cycle of 5%). In most cases, it was possible to identify two distinct halos in the contact area: a dark blue halo, which always represents the central, small area, and a light blue halo, which indicates the larger area formed (Figure 3C). The small area remained relatively constant across all conditions, ranging between 0.3 and 0.39 cm², even as the O₂ concentration in the gas composition increased. The small area matches the contact area between the plasma plume and the agar surface, where we expect to find a higher concentration of ROS during the treatment. For conditions 0.3 and 0.6 using BrD, and condition 0.6 with NwD, only one large zone was identified (see Figure 3A). As a result, the graphs (Figure 3B, C) show data only from this large area for the specified conditions. In these cases, two distinct halos with different colors were not observed; instead, a single dark blue halo formed after exposure to CAP. The results indicate that the large area increases with the addition of O₂ in the gas flow. However, for both devices, after 5 min of treatment under the condition with the highest O₂ concentration (0.6%), it was not possible to distinguish between the two zones, as all halos appeared dark blue, indicating a uniform distribution of ROS throughout the halo. The maximum measured halo areas were 4.84 ± 0.3 cm² with NwD and 6.23 ± 0.4 cm² with BrD. Apparently, with only helium, NwD shows larger halos. It may be related to plume stability and shape: with the NwD, the plume is broader and more stable than with the BrD.
<figure><img src="image_4.png" /><figcaption>FIGURE 3 | (A) Presents the example of the halos formed after the KI-starch exposure to the plasma treatment. The formations of the two zones are highlighted in light blue (large area) and dark blue (small area). The halo's area (cm²) on KI-starch agar plates using (B) BrD and (C) NwD (baseline setting), varying the O₂ concentration from 0% to 0.6%, are presented.</figcaption></figure>

3.3 | Assessment of RONS Concentrations

The RONS measurements showed that the added O₂% signifi-
cantly affects the concentrations of all three RONS species
investigated (O₃, NO, and NO₂). In this sense, BrD in the con-
dition 0% O₂ has a prevalence of NO (38 ppb) compared to the
other chemicals (Figure 4). The 0.16% O₂ condition showed an
increase in NO₂ production (96 ppb). However, for the param-
eters with higher O₂ concentrations (0.3% and 0.6%), O₃ was
more prevalent (13.66 ppm maximum). Some differences were
observed in the NwD (Figure 5), with all concentrations lower
than those in BrD, especially for NO and NO₂. As with the other
device, when the O₂ concentration was 0%, the NO

concentration was the highest among all considered cases (9.5
ppb). Regarding O₃ production, the higher the O₂ concentra-
tion, the higher the measured O₃ concentration, reaching a
maximum of 9.85 ppm.

The concentrations are safe for brief treatment in patients, but long-term exposure must be avoided for patient safety [31]. Considering the safety limit of 48 ppm daily (for a maximum of 8 h of exposure), and that during a 5-min treatment, the patient will be exposed to approximately 70 ppm of ozone, the treatment protocol must be well planned. One suggestion is to perform a maximum of two plasma exposures, with the patient exposed to 140 ppm for two 5-min treatments. This would be a
<figure><img src="image_5.png" /><figcaption>**FIGURE 4** | The concentrations of RONS (O₃, NO, and NO₂) were measured during plasma treatment using BrD. The maximum treatment time was 5 min, with the O₂ concentration between 0% and 0.6% (in each row). The dashed line indicates the moment when the plasma device was deactivated (plasma off). Subsequently, the RONS were measured a few seconds after the plasma was turned off.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**FIGURE 5** | The concentrations of RONS (O₃, NO, and NO₂) were measured during plasma treatment using NwD. The maximum treatment time was 5 min, with the O₂ concentration between 0% and 0.6% (in each row). The dashed line indicates the moment when the plasma device was deactivated (plasma off). Subsequently, the RONS were measured a few seconds after the plasma was turned off.</figcaption></figure>

good strategy once the maximum accumulated exposure to O₃ over 3 days reaches 144 ppm (48 ppm per day). Thus, performing two treatments within 3 days, on alternate days, may be more appropriate clinically and for safety.

Brazilian and French legislation have limitations regarding workers' exposure to the RONS (O₃, NO, NO₂). According to the Brazilian National Air Quality Standards (BNAQS) and the Agence Nationale de Sécurité Sanitaire de l'alimentation, de

l'environnement et du travail (ANSES), the mean limit exposure to O₃ and NO₂ (8 h/day) is 0.1 ppm (100 ppb) for both chemical species. There is no set limit for NO₃ [32]. As this value represents the daily accumulation of 0.1 ppm over 8 h, and the planned plasma treatment protocol was limited to a maximum of 5 min, the safety margin opens the door to the clinical application of both devices for biofilm control, ensuring safety and efficacy. The RONS concentration, as depicted in Figures 4
<table><caption>**TABLE 2** | The concentrations of RONS (O<sub>3</sub>, NO, and NO<sub>2</sub>) were measured during plasma treatment using NwD with the parameters Base, 10x, and 20x.</caption><thead><tr><th rowspan="2"></th><th colspan="3">Accumulated RONS concentration (ppm)</th></tr><tr><th>Base</th><th>10x</th><th>20x</th></tr></thead><tbody><tr><th>O<sub>3</sub></th><td>3.02 ± 0.02</td><td>0.09 ± 0.09</td><td>0.15 ± 0.1</td></tr><tr><th>NO</th><td>4.35 ± 0.06</td><td>0.06 ± 0.001</td><td>0.12 ± 0.003</td></tr><tr><th>NO<sub>2</sub></th><td>8.25 ± 0.1</td><td>9.97 ± 0.1</td><td>34.83 ± 0.8</td></tr></tbody></table>

*Note:* The data presented are the cumulative concentrations measured during the 5-min treatment.

and 5 for both devices, is comfortably within the safe limit for brief treatments.

Regarding the pulse test (Table 2), all treatments conducted with the parameters Base (refer to Table 1) demonstrated accumulated concentrations over a 5-min treatment period of 3.02 ppm (O<sub>3</sub>), 4.35 ppm (NO), and 8.25 ppm (NO<sub>2</sub>). The parameter 10x exhibited accumulated concentrations of 0.09 ppm (O<sub>3</sub>), 0.06 ppm (NO), and 9.97 ppm (NO<sub>2</sub>). Lastly, the 20x parameter recorded accumulated concentrations of 0.15 ppm (O<sub>3</sub>), 0.12 ppm (NO), and 34.83 ppm (NO<sub>2</sub>). It was observed that increasing the number of pulses delivered significantly impacted the levels of reactive oxygen and nitrogen species (RONS) accumulated during the treatment. The Base parameter yielded the highest concentrations of O<sub>3</sub> and NO within the groups, whereas the 10x parameter displayed considerably lower concentrations of these species but a marginally higher concentration of NO<sub>2</sub>. Conversely, the 20x parameter exhibited the highest concentration of NO<sub>2</sub>, which was nearly four times greater than that observed in the other tested parameters.

## 3.4 | Effects of O<sub>2</sub> Addition on Antibacterial Effect

The inhibition halo test results are presented in Figure 6. The treatments were performed for 5 min with BrD and NwD (base setting). The O<sub>2</sub> concentration ranged from 0% to 0.6%, as in the other tests. The inhibition halo under the 0% O<sub>2</sub> condition was the smallest among all conditions when using BrD (0.56 cm<sup>2</sup> for a 5 min treatment). Using NwD, the inhibition halo area was similar among the concentrations up to 0.3% O<sub>2</sub> (reaching values around 0.69 cm<sup>2</sup>).

BrD had no remaining colonies in the inhibition halo area in these conditions. On the other hand, for the NwD, it is possible to spot remaining colonies around the area of inhibition, but much less compared to the same condition (0.1, 0.16, and 0.3% O<sub>2</sub>) when using BrD. Another relevant difference is that, using BrD, the inhibition zones were 0.91, 2.033, and 2.076 cm<sup>2</sup> for the conditions of 0.1%, 0.16%, and 0.3% O<sub>2</sub>, respectively. However, the presence of remaining colonies was higher than observed in NwD. For the 0.6% O<sub>2</sub> condition, the inhibition areas were 2.77 cm<sup>2</sup> (BrD) and 3.23 cm<sup>2</sup> (NwD).

Using BrD, the area of inhibition was well defined, even with isolated colonies. However, using NwD, increasing O<sub>2</sub> in this interval results in a visible reduction in bacterial density around the inhibition halo, but not enough to isolate the colonies. BrD keeps NO<sub>2</sub> generation with the addition of O<sub>2</sub> in the gas flow, except at the highest concentration. Unlike NwD, it mostly delivers O<sub>3</sub> by adding oxygen. Probably the decontamination is

optimized by the presence of both O<sub>3</sub> and NO<sub>2</sub>, as well as by the product of their reaction, N<sub>2</sub>O<sub>5</sub> [33]. The utilization of a CAP jet operating with helium (He) and oxygen (O<sub>2</sub>) has been examined in the literature concerning its impact on *Staphylococcus aureus* biofilms by Xu et al. An oxygen concentration of 0.5% was identified as optimal for biofilm inactivation, aligning with the optimal level observed in the inhibition halo assay conducted in this research. The authors employed mass spectrometry and optical emission spectroscopy to assess the RONS produced by helium and helium/oxygen plasma, noting an increase in atomic oxygen (O) and other oxygen-related ions. These ions may induce strong oxidation and etching processes, potentially resulting in the degradation of organic constituents, such as bacterial cell structures [27].

The behavior of both devices may indicate a transition in the chemical regimen [34]. When only helium (0%) is present, both devices displayed a notable concentration of nitrogen oxides (NO and NO<sub>2</sub>), indicative of an NOx-dominant regimen. As oxygen was incrementally introduced, the NOx concentration was reduced. This likely results from the depletion of NOx due to competitive reactions, without nitrogen compensation [35]. Once the oxygen concentration reaches a sufficiently high level, ozone production increases, particularly above 0.3%, culminating in an ozone-dominant regimen. Temperature also plays an important role in ozone production, as high temperatures are known to decrease O<sub>3</sub> levels [34]. In this sense, the low temperature of the plasma measured in this research may also help maintain O<sub>3</sub> levels.

Wang et al. studied different parameters to achieve either ozone or NOx modes. The power density of the CAP device was utilized to accomplish these modes. High flow rates, longer discharge production times, low power density, and increases in temperature and humidity lead to higher ozone formation (O<sub>3</sub> mode) [36].

In this research, the results showed three distinct behaviors in the inhibition halo test: a completely decontaminated, well-defined circular area with no isolated colonies; a circular inhibited zone with a surrounding zone where microbial density is visibly reduced (indicated by the green arrows in Figure 6), containing isolated colonies; and a well-defined circular inhibited area with isolated colonies. The inhibition halo did not suddenly increase; instead, the surrounding zones, indicated by the green arrows (Figure 6), clearly showed a decrease in bacterial concentration until reaching the measured boundary with a well-defined halo of inhibition. In this context, it appears necessary to have a minimal concentration of RONS for the 5-min treatment, which was not reached under the conditions up to 0.6% using NwD. This behavior was observed using BrD only for the condition with 0.1% O<sub>2</sub>.
<figure><img src="image_7.png" /><figcaption>**FIGURE 6** | Results of *Escherichia coli* inhibition halo test using (A) BrD and (B) NwD. (I) Values of the inhibition halo area (cm²), varying the concentration of added O₂ between 0% and 0.6%. (II) Images of the inhibition halos for each test condition, highlighting the presence of remaining colonies. The green arrows indicate the perimeter around the inhibition zone where the bacteria were not completely inhibited, but the microbial concentration is visibly reduced.</figcaption></figure>
Mestre et al. observed comparable results, using a MHz-jet operating with helium for the inhibition of *E. coli*. Isolated colonies were present at any point in the disinfected area; however, in their case, the border became wider with the longer treatment time (up to 8 min) [37]. The authors suggest that this may occur due to a slight tilt in the plasma jet. This tilt can cause an asymmetric flow pattern, resulting from the gas outlet shape, which impacts the spatial distribution of the after-glow [37].

Pavlovich et al. demonstrated a direct correlation between ozone density and E. coli inactivation in contaminated water treated with DBD plasma, indicating that ozone plays a significant role in bacterial inactivation [38]. In our experiments, it may also be one of the main reasons for the inhibition increasement observed. This is supported by the wider inhibition halos formed for higher O₂ addition, which also led to higher ozone concentration measured during our tests. Once plasma was visible in a smaller area than the inhibition halo observed, especially for higher O₂ concentrations, the reactive species may be carried in the effluent.

Furthermore, Rouillard et al. recently showed—using a cold atmospheric plasma jet operated during parabolic flights—that the plasma plume length and the delivery of reactive species strongly depend on gas composition, especially on O₂ admixture, which directly affects RONS transport and the size of inactivation areas. Their observations confirm that increasing O₂ shortens the plasma plume but enhances the production of long-lived species such as ozone, which can contribute to inactivation at distances beyond the visible plasma reach. This behavior is consistent with our findings, where larger inhibition halos were obtained despite the plasma being visible over a smaller area, particularly at higher O₂ concentrations [39].

Ellerweg et al. studied plasma jet operating with He/O₂ working gas. The authors demonstrated that the concentration of ozone increased with distance from the nozzle when measuring the effluent. The authors also measured the density of atomic oxygen. The results showed a maximum concentration at 0.6% O₂ added to the He/O₂ admixture [40]. Jeong et al. demonstrated that atomic oxygen recombines with O₂ to produce O₃ [41]. This may be the reason for an increase in the inhibition halo, resulting from the increased production of O₃ in the plasma effluent.

The inhibition halo test using NwD to evaluate the effect of the number of pulses during treatment is shown in Figure 7. When testing the base protocol and the 10x condition during a 5-min treatment (Figure 7A), the 10x increase in pulses delivered over the same time period increases the inhibition halo area from 0.59 cm² (Base) to 2.76 cm² (10x). The second test (Figure 7B) evaluated whether, using parameter 10x, it would be possible to decrease the treatment time by 10fold (from 300 s to 30 s) and achieve a similar inhibition outcome. As a result, both configurations indeed showed equivalent results of 0.59 and 0.60 cm² (base and 10x, respectively). This means that, when delivering 150 000 pulses at different times, the same halo-inhibiting area was achieved in both conditions, allowing for a much shorter treatment time (from 300 s to 30 s) without compromising performance.

<figure><img src="image_8.png" /><figcaption>**FIGURE 7** | Results from the *E. coli* inhibition halo test using NwD showed that the number of pulses delivered during treatment varied. A, B, and C are different test conditions with varying numbers of pulses and treatment times. Different lowercase letters indicate statistically significant group differences (*p* < 0.05).</figcaption></figure>
After a 5-min treatment using the 10x protocol, the inhibition halo area was 2.76 cm². The third test (Figure 7C) aimed to evaluate whether we could achieve a similar result in half the time (150 s) by increasing the parameter to 20x (delivering a total of 150 000 pulses over the entire treatment). The results indicate that the inhibition halo achieved at 10x was 2.76 cm², and at 20x, 2.17 cm². The values are closer when considering the standard deviation. However, the inhibition halo is narrower than the base parameter. With the 20x configuration, the inhibition halo obtained was almost 4 times broader in the half-time treatment (150 s). A correlation was observed between the dark blue area and complete central bacterial inhibition. In contrast, the light blue area observed resulted in a partial inhibition halo, with remaining colonies. This observation supports our hypothesis that depositing a KI-starch gel into the T-tube and exposing it to plasma treatment can be a valuable alternative for assessing the CAP jet's potential for bacterial decontamination. In this sense, the correlation between the dark blue color and the assessment of RONS distribution within the T-tube can provide information on their antibacterial activity.

## 3.5 | Assessment of Reactive Oxygen Species (ROS) Distribution Inside the T-Tube

The formation of a thin layer of KI-starch agar within the T-tubes represents a qualitative approach to investigating the delivery of reactive species within the tubes, an innovative strategy for controlling biofilms. During the test, the plasma plume was generated inside the tube as shown in Figure 8.

Figure 9 presents the images of the tubes exposed to the plasma treatment for 5 min using the best configurations studied. The NwD 0% O₂ configuration showed local interaction of the reactive species at the interface between the plasma plume and the agar surface, which did not spread throughout the tube. The NwD 10x configuration with no oxygen admixture showed higher ROS production, as observed in the previous tests, but did not cover the entire tube length. The NwD 0.6% O₂ configuration was the most efficient for delivering ROS throughout the T-tube. As discussed in the previous sections, even if we could not find a strict relation between the KI-starch halo formation and the E. coli inhibition halo, all the regions that

<figure><img src="image_9.png" /><figcaption>**FIGURE 8** | Cold atmospheric plasma plume generated inside a T-tube. The capillary was connected to the vertical part of the tube, also called the short laryngeal part.</figcaption></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**FIGURE 9** | Photography of the T-tubes with deposited KI-starch on the inner walls after plasma exposure to the best parameters studied up to now with the NwD.</figcaption></figure>

presented a dark blue area in the KI-starch test also showed at least a partial inhibition halo. In this way, it can be concluded that this condition offers a promising alternative to bacterial control within the T-tubes and warrants further exploration.

## 3.6 | Measurements of Temperature

The temperature measurement was relevant for confirming that bacterial decontamination was not due to thermal stress and for understanding the influence of varying the number of pulses from NwD across conditions and how this would affect the target temperature during the treatment (Figure 10). Understanding this is important when considering the feasibility of this treatment in clinics. The BrD operating with only He was also assessed, showing a target temperature of around 20.9°C during the treatment. NwD also has a similar temperature when running with only He in the base protocol, around 20.3°C. As the number of pulses increased, the temperatures increased in the 10x and 20x conditions, reaching 26.7°C and 48.5°C, respectively. The results indicate that the plasma device, in
<figure><img src="image_13.png" /><figcaption>**FIGURE 10** | Plasma jet temperature on target assessment, with both plasma devices (BrD and NwD) for the base parameter, and investigation on the increment of pulses (10x and 20x).</figcaption></figure>

terms of temperature, is safe for clinical application. For a direct plasma jet application in patients, the maximum temperature is around 40°C [4]. In this study, the plasma is intended to be applied within the T-tube, not directly to the patient. The reported temperatures are within a safe range for the application. Also, the treatment can be performed using different protocols, such as splitting the total 5-min treatment into 60-second rounds interspersed with a resting period. It can be an approach to avoid high temperatures during long-term therapy. Another approach would be to increase the helium flow rate, which can lead to the effluent and target cooling. Furthermore, as different O₂ concentrations were added to the gas flow, shorter treatment times may be a promising alternative.

## 4 | Conclusion

This study investigated various compositions of working gases by adjusting the O₂ concentration within the base He gas flow. Additionally, different pulse numbers were assessed during the treatment process to optimize procedures for specific applications. Furthermore, the relationship between the distribution of reactive oxygen and nitrogen species (RONS) on the agar surface, their concentrations during treatment, and their antibacterial effectiveness was examined. Generally, changes in the working gas composition affect the dominant reactive species and their levels throughout plasma treatment. The treatment area can also be adjusted based on the gas composition and the number of pulses used. However, adding O₂ to the working gas decreases the homogeneity of the treatment, making complete microbial eradication more difficult. Nonetheless, the broader range of RONS delivery could have important implications for future clinical applications. The KI-starch assay on agar plates did not precisely predict the size of the inhibition halos observed in the E. coli test; however, the inhibition zone was consistently contained within the KI-starch-affected region. Therefore, the KI-starch-affected area can serve as an effective upper limit for the inhibition zone. Overall, the KI-starch

method remains a useful preliminary screening tool for identifying the most promising approaches.

All data obtained from this research are relevant to the use of plasma jets for treating biofilms within endotracheal prostheses. The treatment of KI-starch agar deposited in T-tubes was a preliminary, qualitative assessment that showed the potential of plasma jets, under proper conditions and parameters, as a promising clinical alternative.

## Acknowledgments

This work was supported by the São Paulo State Research Foundation (FAPESP), grant numbers 2019/05856-7, 2021/02680-5, and 2023/02436-2; the Coordination for the Improvement of Higher Education (CAPES); and the Brazilian National Council for Scientific and Technological Development (CNPq). The authors also acknowledge COST Action, Action CA20114 PlasTHER "Therapeutic Applications of Cold Plasmas," supported by COST (European Cooperation in Science and Technology). The Article Processing Charge for the publication of this research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (ROR identifier: 00x0ma614).

## Conflicts of Interest

The authors declare no conflicts of interest.

## Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

## References

1. B. J. Bibas, P. F. G. Cardoso, M. Salati, et al., "Health-Related Quality of Life Evaluation in Patients With Non-Surgical Benign Tracheal Stenosis," *Journal of Thoracic Disease* 10 (2018): 4782-4788.

2. K. Ding, Y. Wang, S. Liu, S. Wang, and J. Mi, "Preparation of Medical Hydrophilic and Antibacterial Silicone Rubberviasurface Modification," *RSC Advances* 11 (2021): 39950-39957.

3. K. Mazhar, M. Gunawardana, P. Webster, et al., "Bacterial Biofilms and Increased Bacterial Counts Are Associated With Airway Stenosis," *Otolaryngology-Head and Neck Surgery* 150 (2014): 834-840.

4. C. Piazza, M. Filauro, F. G. Dikkers, et al., "Long-Term Intubation and High Rate of Tracheostomy in COVID-19 Patients Might Determine an Unprecedented Increase of Airway Stenoses: A Call to Action From the European," *Laryngological Society European Archives of Oto-Rhino-Laryngology* 278 (2021): 1-7.

5. R. Ershadi, S. Rafieian, J. Sarbazzadeh, and M. Vahedi, "Tracheal Stenosis Following Mild-to-Moderate COVID-19 Infection Without History of Tracheal Intubation: A Case Report," *General Thoracic and Cardiovascular Surgery* 70 (2022): 303-307.

6. F. Mattioli, A. Marchioni, A. Andreani, G. Cappiello, M. Fermi, and L. Presutti, "Post-Intubation Tracheal Stenosis in COVID-19 Patients," *European Archives of Oto-Rhino-Laryngology* 278 (2021): 847-848.

7. C. F. Gervasio, G. Averono, L. Robiolio, et al., "Tracheal Stenosis After Tracheostomy for Mechanical Ventilation in COVID-19 Pneumonia - A Report of 2 Cases From Northern Italy," *American Journal of Case Reports* 21 (2020): e926731.

8. S. A. R. Nouraei, M. A. Petrou, S. R. Prem, et al., "Bacterial Colonization of Airway Stents a Promoter of Granulation Tissue Formation Following Laryngotracheal Reconstruction," *American Society for Head and Neck Surgery* 132 (2006): 1086-1090.
9. B. J. Bibas, R. M. Terra, A. L. Oliveira Junior, et al., "Predictors for Postoperative Complications After Tracheal Resection," *Annals of Thoracic Surgery* 98 (2014): 277-282.

10. R. Ershadi, S. Rafieian, J. Sarbazzadeh, and M. Vahedi, "Tracheal Stenosis Following Mild-to-Moderate COVID-19 Infection Without History of Tracheal Intubation: A Case Report," *General Thoracic and Cardiovascular Surgery* 70 (2022): 303-307.

11. S. Bekeschus, F. Saadati, and S. Emmert, "The Potential of Gas Plasma Technology for Targeting Breast Cancer," *Clinical and Translational Medicine* 12 (2022): e1022.

12. S. Duarte and B. H. D. Panariello, "Comprehensive Biomedical Applications of Low Temperature Plasmas," *Archives of Biochemistry and Biophysics* 693 (2020): 108560.

13. A. K. Martusevich, A. V. Surovegina, I. V. Bocharin, V. V. Nazarov, I. A. Minenko, and M. Y. Artamonov, "Cold Argon Atmospheric Plasma for Biomedicine: Biological Effects, Applications and Possibilities," *Antioxidants* 11 (2022): 1262.

14. L. Miebach, B. Poschkamp, J. van der Linde, and S. Bekeschus, "Medical Gas Plasma—A Potent ROS-Generating Technology for Managing Intraoperative Bleeding Complications," *Applied Sciences* 12 (2022): 3800.

15. A. C. Borges, G. M. G. Lima, T. M. C. Nishime, A. V. L. Gontijo, K. G. Kostov, and C. Y. Koga-Ito, "Amplitude-Modulated Cold Atmospheric Pressure Plasma Jet for Treatment of Oral Candidiasis: In Vivo Study," *PLoS One* 13 (2018): e0199832.

16. K. G. Kostov, A. C. Borges, C. Y. Koga-Ito, T. M. C. Nishime, V. Prysiazhnyi, and R. Y. Honda, "Inactivation of *Candida albicans* by Cold Atmospheric Pressure Plasma Jet," *IEEE Transactions on Plasma Science* 43 (2015): 770-775.

17. F. do Nascimento, A. da Graça Sampaio, N. V. M. Milhan, et al., "A Low Cost, Flexible Atmospheric Pressure Plasma Jet Device With Good Antimicrobial Efficiency," *IEEE Transactions on Radiation and Plasma Medical Sciences* 8 (2024): 307-322.

18. K. Trebulová, I. Orel, J. M. Pouvesle, et al., "Antimycotic Effects of the Plasma Gun on the Yeast *Candida glabrata* Tested on Various Surfaces," *Plasma Processes and Polymers* 21 (2024): e2400057.

19. O. Bastin, M. Thulliez, J. Servais, et al., "Optical and Electrical Characteristics of an Endoscopic DBD Plasma Jet," *Plasma Medicine* 10 (2020): 71-90.

20. H. Decauchy, A. Pavy, M. Camus, L. Fouassier, and T. Dufour, "Cold Plasma Endoscopy Applied to Biliary Ducts: Feasibility Risk Assessment on Human-Like and Porcine Models for the Treatment of Cholangiocarcinoma," *Journal of Physics D: Applied Physics* 55 (2022): 455401.

21. F. Theinkom, L. Singer, F. Cieplik, et al., "Antibacterial Efficacy of Cold Atmospheric Plasma Against Enterococcus faecalis Planktonic Cultures and Biofilms In Vitro," *PLoS One* 14 (2019): e0223925.

22. F. İbiş and U. K. Ercan, "Inactivation of Biofilms in Endotracheal Tube by Cold Atmospheric Plasma Treatment for Control and Prevention of Ventilator-Associated Pneumonia," *Plasma Processes and Polymers* 17 (2020): e2000065.

23. G. de Morais Gouvêa Lima, C. F. L. Carta, A. C. Borges, et al., "Cold Atmospheric Pressure Plasma Is Effective Against P. gingivalis (HW24D-1) Mature Biofilms and Non-Genotoxic to Oral Cells," *Applied Sciences* 12 (2022): 7247.

24. G. Jungbauer, L. Favaro, S. Müller, A. Sculean, and S. Eick, "The In-Vitro Activity of a Cold Atmospheric Plasma Device UTI-Lizing Ambient Air Against Bacteria and Biofilms Associated With Periodontal or Peri-Implant Diseases," *Antibiotics (USSR)* 11 (2022): 752.

25. Q. Wang, N. Lavoine, and D. Salvi, "Cold Atmospheric Pressure Plasma for the Sanitation of Conveyor Belt Materials: Decontamination Efficacy Against Adherent Bacteria and Biofilms of Escherichia coli and

Effect on Surface Properties," *Innovative Food Science & Emerging Technologies* 84 (2023): 103260.

26. S. Das, V. P. Gajula, S. Mohapatra, G. Singh, and S. Kar, "Role of Cold Atmospheric Plasma in Microbial Inactivation and the Factors Affecting Its Efficacy," *Health Sciences Review* 4 (2022): 100037.

27. Z. Xu, J. Shen, C. Cheng, S. Hu, Y. Lan, and P. K. Chu, "In Vitro Antimicrobial Effects and Mechanism of Atmospheric-Pressure He/O2 Plasma Jet on Staphylococcus aureus Biofilm," *Journal of Physics D: Applied Physics* 50 (2017): 105201.

28. A. Baz, A. Bakri, M. Butcher, et al., "Staphylococcus aureus Strains Exhibit Heterogenous Tolerance to Direct Cold Atmospheric Plasma Therapy," *Biofilm* 5 (2023): 100123.

29. V. Udachin, S. Dahle, and R. Fink, "Dielectric Barrier Discharge Air Plasma as a Comprehensive Disinfection Approach to E. coli and S. aureus Biofilm Management," *Physica Scripta* 100 (2025): 025015.

30. D. M. Silva, F. Do Nascimento, N. V. M. Milhan, et al., "Cold Atmospheric Helium Plasma in the Post-COVID-19 Era: A Promising Tool for the Disinfection of Silicone Endotracheal Prostheses," *Microorganisms* 12 (2024): 130.

31. M. S. Mann, R. Tiede, K. Gavenis, et al., "Introduction to DIN-Specification 91315 Based on the Characterization of the Plasma Jet Kinpen® Med," *Clinical Plasma Medicine* 4 (2016): 35-45.

32. B. Siciliano, G. Dantas, C. da Silva, and G. Arbilla, "The Updated Brazilian National Air Quality Standards: A Critical Review," *Journal of the Brazilian Chemical Society* 31 (2020): 523-535.

33. S. Park, W. Choe, and C. Jo, "Interplay Among Ozone and Nitrogen Oxides in Air Plasmas: Rapid Change in Plasma Chemistry," *Chemical Engineering Journal* 352 (2018): 1014-1021.

34. S. Park, W. Choe, and C. Jo, "Interplay Among Ozone and Nitrogen Oxides in Air Plasmas: Rapid Change in Plasma Chemistry," *Chemical Engineering Journal* 352 (2018): 1014-1021.

35. J. Kim, H. Lee, S. C. Huh, et al., "Competitive Formation of NO, NO₂, and O₃ in an Air-Flowing Plasma Reactor: A Central Role of the Flow Rate," *Chemical Engineering Journal* 468 (2023): 143636.

36. Z. Wang, L. Wang, P. Zhang, et al., "Time-Sharing Synergy of NOx Mode and O₃ Mode Air Discharges for Efficient and Pollution-Free Disinfection," *Plasma Processes and Polymers* 21 (2024): e2400083.

37. E. Mestre, I. Orel, D. Henze, et al., "Comparison of CO Production and Escherichia Coli Inactivation by a kHz and a MHz Plasma Jet," *Plasma Processes and Polymers* 21 (2024): e2300182.

38. M. J. Pavlovich, H. W. Chang, Y. Sakiyama, D. S. Clark, and D. B. Graves, "Ozone Correlates With Antibacterial Effects From Indirect Air Dielectric Barrier Discharge Treatment of Water," *Journal of Physics D: Applied Physics* 46 (2013): 145202.

39. A. Rouillard, P. Escot Bocanegra, A. Stancampiano, et al., "Demonstration for Cold Atmospheric Pressure Plasma Jet Operation and Antibacterial Action in Microgravity," *NPJ Microgravity* 10 (2024): 74.

40. G. Willems, J. Golda, D. Ellerweg, et al., "2019 Corrigendum: Characterization of the Effluent of a He/O₂ Micro-Scaled Atmospheric Pressure Plasma Jet by Quantitative Molecular Beam Mass Spectrometry," *New Journal of Physics* 12, no. 013021 (2010): 013021, https://doi.org/10.1088/1367-2630/12/1/013021.

41. J. Y. Jeong, J. Park, I. Henins, et al., "Reaction Chemistry in the Afterglow of an Oxygen-Helium, Atmospheric-Pressure Plasma," *Journal of Physical Chemistry A* 104 (2000): 8027-8032.