## 4 Discussion  
The aim of this study was to design a concept for a contact-free spacer to visualize the effective treatment area of the cold atmospheric-pressure plasma jet kINPen MED and to detect and control the treatment distance. This should enable a contactless plasma treatment with an unobstructed, illuminated view over the effective area and free energy exchange with the surrounding air. In order to realize the visualization of an effective treatment area, a variety of measurements were performed to verify the assumption of an impact of the treatment angle on the effective area. It was confirmed that the effective area was strongly dependent on the treatment angle. Thus, a sensor arrangement that visualizes the effective area has to consider the treatment angle. Furthermore, the individual effective areas of ROS, (V)UV and inhibition zone assays were not congruent. This indicated specific distribution properties. The plasma effective zone was analyzed by three test settings, the starch-potassium iodide test, the (V)UV determination and the inhibition zone assay. In the latter case _S. aureus_ (DSM 799/ATCC 6538) was used as test microorganism. The starch-potassium iodide test was used to provide an alternative fast and easy method for the determination of the plasma effective zone via ROS surface contact distribution without the utilization of biological samples, such as microorganisms. The biological effective area for _S. aureus_ was larger with increasing incidence angle. Thus, it is necessary to know and visualize the effective area to ensure an optimal treatment of the targeted surface, e.g., a wound. The antimicrobial and ROS analyses resulted in circular zones when the kINPen(r) MED-\(\beta\) was applied vertically and in elliptical/comet-like zones after tilting of the device. Under the chosen treatment conditions (45 s and 0\({}^{\circ}\), 25\({}^{\circ}\), 50\({}^{\circ}\)), the inhibition zone was between the secondary and the peripheral zone of the ROS formation regarding width and similar to the peripheral zone for the length of ROS formation. However, the total area of the inhibition zone increased, whereas ROS decreased (at least by 9%) with rising treatment angle. This supports the assumption that ROS is one main factor for antimicrobial efficacy, but not the only one. This may also be explained by the selectivity of the starch-potassium iodide plates. In this test, the proof of plasma efficacy was shown by the reactivity of the ROS with the surface of the starch-potassium iodide plates using different angles of the plasma source. The maximum ROS density was measured directly in the visible effluent [6; 31]. In this area, a violet effluent was formed around the blue argon part, whereby the effluent consisted mainly of an ionized ROS environment [32; 33]. Additionally, it is well known that a low leakage current occurs at the contact area of the effluent with the starch-potassium iodide plates [34]. This might result in the formation of polyiodide and thereby in a dark brown to black discoloration of the primary zone that is not caused solely by a ROS-based oxidation process. The ROS formed around the plasma effluent should be transported with the argon gas flow to the surface and spread according to the flow direction, which leads to a comet-shaped effective area. The secondary zone is supposed to be the result of less intense short-lived ROS. The density of the ROS decreased with increasing distance from the plasma zone. This is caused by reactions with ambient air, diffusion, turbulent gas flows and other interactions with the environment while being countered by argon metastable recombination processes releasing energy and inducing a complex plasma chemistry even in the afterglow [35]. We hypothesized that this resulted in an extensive peripheral zone.