# Water Modification by Cold Plasma Jet with Respect to Physical and Chemical Properties
## 2 Experimental Setup and Materials
### ROS Detection in the Liquids  
Apart from the basic liquid characterization, before and after the plasma treatment, various reagents, standard, and special solutions are used for the detection of principal chemical radicals in both the DDW and the PBS. The materials and the procedures are given below in detail. Reagents  
* Hydroethidine (HE; Fluka, Buchs, Switzerland; cat. no. 37291). * Dimethyl sulfoxide (DMSO, 99.9% or 14 M; ChemLab, Zedelgem, Belgium; cat. no. CL00.0422). * Hydrochloric acid (HCl, 37% w/w or 12 M; ChemLab, Zedelgem, Belgium; cat. no. CL00.0310). * 2-hydroxy terephthalate (2-OH-TPA, 98%; TCI, Portland, USA; cat. no. H1385). * 2-hydroxy ethidium (2-OH-E\({}^{+}\), synthesized as described elsewhere [33]). * Sodium hydroxide (NaOH, pellets; Merck, New Jersey, USA; cat. no. 106498). * Phosphate Buffered Saline (PBS powder, for preparing 1 L, pH 7.4; Sigma-Aldrich, Burlington, MA, USA; P3813). * Terephthalalic acid (TPA, 98+%; Alfa Aesar, Kandel, Germany; cat. no. A12527). * DDW, purified by a Milli-Q system (Millipore, Massachusetts, USA). * Acetonitrile (ACN, HPLC grade; Sigma-Aldrich, Burlington, MA, USA; cat. no. 34851). * Chloroform (CHCl\({}_{3}\); Merck, New Jersey, USA; cat. no. 1.02445). * Methanol (MetOH, \(\geq\)99.9%; Merck, New Jersey, USA; cat. no. 106009). Standard solutions  
* 5 mM TPA, pH 7: Dissolve 4.15 mg TPA in 1 mL 0.1 M NaOH and ddH\({}_{2}\)O is added to 5 mL final volume. Adjust pH to 7, by dropwise addition of 0.5 M HCl. Long storage at \(-\)20 \({}^{\circ}\)C.
* 1 M DMSO: prepare 1 mL by mixing 71.4 \(\mu\)L 14 M DMSO with 928.6 \(\mu\)L DDW. * 0.4 M HCl: prepare 2 mL by mixing 66.7 \(\mu\)L 12 M HCl with 1933.3 \(\mu\)L DDW. * 5 M NaOH: dissolve 0.4 g in DDW to final 2 mL. * 20 mM HE: dissolve 6.3 mg HE in 1 mL 0.4 M HCl. Long storage at \(-\)20 \({}^{\circ}\)C.
* PBS, pH 7: mix the content of a PBS bag with 1 L DDW. * 1 M DMSO in PBS, pH 7: prepare 1 mL by mixing 71.4 \(\mu\)L 14 M DMSO with 928.6 \(\mu\)L PBS. * 5 mM 2-OH-TPA, pH 7: dissolve 1.4 mg HTPA in 0.3 mL 0.1 M NaOH and add DDW to final 1.5 mL. The pH is neutralized to 7, by addition of about 10 \(\mu\)L 1 M HCl. Long storage at \(-\)20 \({}^{\circ}\)C.
* 0.2 mM 2-OH-E\({}^{+}\) (in 0.4 M HCl) prepared from synthetic 2-OH-E\({}^{+}\) as described elsewhere [33]. Long storage at \(-\)20 \({}^{\circ}\)C.
* 50 mM Pi buffer, pH 7.8: dissolve 0.45 g Na\({}_{2}\)HPO\({}_{4}\).2H\({}_{2}\)O in about 48 mL DDW, final 50 mL DDW, adjust to pH 7.8 (dropwise with 1 M HCl) and bring to final 50 mL with DDW. TPA-based ROS detection solutions: for \({}^{\bullet}\)OH and H\({}_{2}\)O\({}_{2}\)  
* 100 \(\mu\)M TPA, pH 7: prepare 8 mL by mixing 0.16 mL 5 mM TPA with 7.84 mL DDW, and if needed adjust pH to 7. * 100 \(\mu\)M TPA + 50 mM DMSO, pH 7: prepare 8 mL by mixing 0.16 mL 5 mM TPA with 0.4 mL 1 M DMSO and 7.44 mL DDW and, if needed, adjust pH to 7. * 100 \(\mu\)M TPA + 50 mM DMSO in PBS, pH 7: prepare 8 mL for each experiment by mixing 0.16 mL 5 mM TPA with 0.4 mL 1 M DMSO and 7.44 mL PBS and, if needed, adjust pH to 7. 100 \(\upmu\)M HE, pH 7: prepare 8 mL for each experiment by mixing 40 \(\upmu\)L 20 mM HE with 7.96 mL DDW. Adjust pH to 7 by dropwise addition of 5 M NaOH. - 100 \(\upmu\)M HE in PBS, pH 7: prepare 8 mL for each experiment by mixing 40 \(\upmu\)L 20 mM HE with 7.96 mL PBS and, if needed, adjust pH to 7. Detection of \({}^{\bullet}\)OH and H\({}_{2}\)O\({}_{2}\)  
\({}^{\bullet}\)OH and H\({}_{2}\)O\({}_{2}\) are measured in the 8-mL ROS (detecting) solutions containing TPA (specific probe for \({}^{\bullet}\)OH), TPA+DMSO (DMSO is specific scavenger for \({}^{\bullet}\)OH; serves as plasma-generated \({}^{\bullet}\)OH elimination control), TPA in PBS, and TPA+DMSO in PBS, which are each placed in 10-mL glass beakers.