# Anti-Bacterial Action of Plasma Multi-Jets in the Context of Chronic Wound Healing
## 2 Materials and Methods  
### Plasma Jet Devices  
This work is based on the use of non-thermal plasma devices for in vitro applications and a pilot study. Based on our know-how at GREMI, the plasma gun technology [42; 43] that was used in this work needed to meet two main requirements: to develop plasma jets while keeping the temperature of the target sample below 40 \({}^{\circ}\)C, and to afford the best strategy to expose large contaminated surfaces during short time treatments. With regard to the treatment of infected and chronic wounds, the typical surfaces to be exposed are of a few to tens of square centimeters large and exhibit structured three dimensional topologies, i.e., they do not necessarily consist of flat targets. Thus, the whole surface exposure should be performed with likely significant variations of the distance between the plasma jet and the sample and the full time treatment should not exceed a few minutes. The first option, recently reported in [44], is to expose large surfaces to a room temperature single plasma jet powered with high frequency (typically 10 to 20 kHz with our device) voltage pulses of microseconds in duration. The tuning of the voltage peak amplitudes, gas flow rates, and the consideration of the target electrical conductivity features was demonstrated to allow for the controlled exposure of a surface with a typical diameter of 2 cm. Nevertheless, the larger surface exposure was not easy to achieve without an additional displacement of the plasma plume, and this device was to be operated at voltages ranging from 10 to 20 kV. The second option consisted in the operation of so-called multi-jets generated from a single primary jet [43; 45; 46]. These multi-jets were used for the in vitro study performed in this work with two applicators: the one delivering multi-jets in ambient air leading to a multi-spot impact, and the other using the same device but with an additional sleeve leading to helium accumulation over the target, while not being fully airtight to maintain reactive oxygen and nitrogen species generation. This second applicator results in plasma delivery as a combination of multi-spots and a diffuse plasma generated in between the individual jets. These two multi-jet devices are called \"multi-spot\" and \"diffuse mode\" applicators in the following analysis. Figure 1 presents the schematics of these two devices. As shown in Figure 1, any of the two plasma applicators was connected through a flexible silicone tube inside which high purity (99.999%) helium was flushed at 4 L/min. The pulse power supply generates voltage pulses of 4 \(\upmu\)s FWHM, having a peak amplitude of +14 kV and delivered at a 2 kHz repetition rate. These voltage pulses were delivered via an isolated copper wire inserted all along the silicone tube to the tip located at the inlet of the shower-like multi-jets applicator schematically documented in Figure 1. This remote plasma jet generation, including a flushed gas and high voltage wire inside a flexible tube, was previously reported for endoscopic application [47] and multi-jets generation [48]. The shower-like applicator consists of a dielectric (Delrin (r)) assembly having an inlet volume (50 mm in diameter, 20 mm height) and an outlet part equipped with 52 channels (0.8 mm in diameter, 10 mm height). These 52 channels are distributed along four rings of 8, 18, 28 and 38 mm in diameter and having 4, 12, 12 and 24 holes, respectively. The discharge is ignited at the tip of the high voltage wire as a primary helium plasma jet. This primary plasma jet propagates in the inlet volume of the shower-like applicator before splitting into secondary helium jets generated in the channels of the outlet section of the applicators [46]. With the multi-spot plasma applicator, all of the secondary jets distributed along the 8, 18, 28 and 38 mm diameter rings were delivered simultaneously. The plasma that was in contact with the sample is associated with ionization wave propagation in the helium gas flow at the outlet of the capillary.