Non-Thermal Plasma Technologies for Plant Virus Inactivation: Sources, Mechanisms, and Practical Applications.

Plasma-activated water (PAW) is receiving much attention in biomedical applications because of its reported potent bactericidal properties. Reactive oxygen and nitrogen species (RONS) that are generated in water upon plasma exposure are thought to be the key components in PAW that destroy bacterial and cancer cells. In addition to developing applications for PAW, it is also necessary to better understand the RONS chemistry in PAW in order to tailor PAW to achieve a specific biological response. With this in mind, we previously developed a UV–vis spectroscopy method using an automated curve fitting routine to quantify the changes in H2O2, NO2−, NO3− (the major long-lived RONS in PAW), and O2 concentrations. A major advantage of UV–vis is that it can take multiple measurements during plasma activation. We used the UV–vis procedure to accurately quantify the changes in the concentrations of these RONS and O2 in PAW. However, we have not yet provided an in-depth commentary of how we perform the curve fitting procedure or its implications. Therefore, in this study, we provide greater detail of how we use the curve fitting routine to derive the RONS and O2 concentrations in PAW. PAW was generated by treatment with a helium plasma jet. In addition, we employ UV–vis to study how the plasma jet exposure time and treatment distance affect the RONS chemistry and amount of O2 dissolved in PAW. We show that the plasma jet exposure time principally affects the total RONS concentration, but not the relative ratios of RONS, whereas the treatment distance affects both the total RONS concentration and the relative RONS concentrations.

Plasma-activated water (PAW) is widely recognized as a promising anticancer agent owing to its abundance of reactive oxygen and nitrogen species (RONS), which selectively target cancer cells. However, the clinical translation of PAW has been hindered by the relatively low concentration and short half-lives of its RONS components. Herein, this Letter proposes a promising strategy for cancer therapy via utilizing plasma-activated micro-nano bubbles water (MNBs-PAW). Compared with conventional PAW, MNBs-PAW exhibits significantly enhanced concentrations and prolonged half-lives of aqueous RONS. Notably, while the H2O2 concentration shows a slight decrease, the levels of NO2−, ONOO−/O2−, OH, and 1O2 are substantially elevated. Furthermore, the half-lives of short-lived species (ONOO−/O2−, OH, and 1O2) are extended by factors of 4, 4, and 2, respectively. These improvements are attributed to the introduced micro-nano bubbles enhancing the gas–liquid mass transfer efficiency of gaseous RONS and prolonging the residence time of aqueous RONS through their unique interface effects. The elevated RONS levels and their synergistic redox reactions induce a significant accumulation of intracellular ROS levels within A549 cancer cells (predominantly O2− and OH), indicating that MNBs-PAW possesses substantially superior anticancer efficacy to conventional PAW. This study not only introduces an innovative approach for PAW preparation but also highlights a promising direction for clinical cancer therapy.

Nonthermal biocompatible plasma (NBP) sources operating in atmospheric pressure environments and their characteristics can be used for plasma bioscience, medicine, and hygiene applications, especially for COVID-19 and citizen. This review surveyed the various NBP sources, including a plasma jet, micro-DBD (dielectric barrier discharge) and nanosecond discharged plasma. The electron temperatures and the plasma densities, which are produced using dielectric barrier discharged electrode systems, can be characterized as 0.7 ~ 1.8 eV and (3–5) × 1014–15 cm−3, respectively. Herein, we introduce a general schematic view of the plasma ultraviolet photolysis of water molecules for reactive oxygen and nitrogen species (RONS) generation inside biological cells or living tissues, which would be synergistically important with RONS diffusive propagation into cells or tissues. Of the RONS, the hydroxyl radical [OH] and hydrogen peroxide H2O2 species would mainly result in apoptotic cell death with other RONS in plasma bioscience and medicines. The diseased biological protein, cancer, and mutated cells could be treated by using a NBP or plasma activated water (PAW) resulting in their apoptosis for a new paradigm of plasma medicine.

Despite notable advances in anticancer drug development, their manufacture and use pose environmental and health risks due to toxic byproducts, drug residue contamination, and cytotoxicity to normal cells. Therefore, developing cost-effective anticancer treatments with fewer toxic side effects and higher selectivity is essential to the advancement of highly effective anticancer therapies. Plasma-activated water (PAW) offers a green alternative to conventional chemical treatments as it reverts to water within days. However, the limited duration and dose of reactive oxygen and nitrogen species (RONS) in acidified PAW restrict its clinical deployment and the full understanding of their mechanism. In this study, we propose alkaline PAW as an innovative enhancement of the RONS technology. The alkaline PAW generated markedly superior RONS, with about 10 times higher levels of NO2-, H2O2, and ONOO-/O2•- than acidic PAW. The possible RONS generation pathways in alkaline PAW are analyzed by scavengers. In conventional acidic PAW, 70% of the H2O2 concentration is contributed by •OH but only about 20% in alkaline PAW. ONOO- is mainly formed through the reaction of O2•- with NO in alkaline pH, while in acidic PAW, it mainly forms from NO2- and H2O2. The results unveiled the synergistic and formidable anticancer effects of alkaline PAW against cancer cells, typified by an increase in intracellular ROS/RNS levels. Furthermore, alkaline PAW injection also effectively prevented xenograft tumor growth in mice. We systematically investigated this high-dose anticancer solution without using noble gases, toxic reagents, or extra energy consumption and successfully demonstrated the possibility of alkaline PAW being an effective and environmentally friendly therapeutic technology. The activity is closely linked to the RONS dose, and the generation pathway provides much-needed insight into the fundamental aspects of PAW chemistry required for the optimization of the biochemical activity of PAW.

Cold plasma-liquid interaction becomes a growing interdisciplinary area of research involving plasma physics, fluid science, and chemistry. Plasma-liquid interaction has gained more interest over the last many years due to its potential applications in different fields. Cold atmospheric plasma jet is an emerging technology for surface drinking water treatment to improve quality and surface modification that is chemical-free and eco-friendly. Cold plasma treatment of water samples results in changes in turbidity, pH, and conductivity and in the formation of reactive oxygen and nitrogen species (RONS). As a result, plasma-activated water has a different chemical composition than water and can serve as an alternative technique for microbial disinfection. CAPJ has been generated by a high voltage 5 kV and a high frequency 19.56 kHz power supply. The discharge has been characterized by an optical method. To characterize the cold atmospheric pressure argon plasma jet, discharge plume temperature, and electron rotational and vibrational temperature have been determined. Cold atmospheric argon plasma jet produced at atmospheric condition contains high energetic electrons, ions, UV radiation, reactive oxygen, and nitrogen species named as cold plasma which has a wide range of applications in the biomedical industry, as well as in water treatment. Nowadays, researches have been carried out on ozonation through plasma jet interaction with surface drinking water. In this paper, we compare the change in physical and chemical parameters of surface water used for drinking purposes. The significant change in the physical parameters such as pH, turbidity, and electrical conductivity was studied. In addition, the significant changes in the concentration and absorbance of nitrate, ferrous, and chromium ions with respect to treatment time were studied. Our results showed that plasma jet interaction with surface drinking water samples can be useful for the improvement of water quality and an indicator for which reactive species play an important role in plasma sterilization.

Plasma-activated water (PAW) has emerged as a platform for sterilizing fungal pathogens. In this study, we investigated the influence of PAW on black melanized spores of Aspergillus brasiliensis to explore the mechanism of fungal spore inactivation. PAW was prepared by activating deionized water with a nonthermal atmospheric pressure air plasma jet (soft plasma jet). The concentrations of H2O2 and NOx in the PAW treated by the soft plasma jet for 3 min were 50 μM and 1.8 mM, respectively, and the pH of the PAW was 3.10. The reactive oxygen and nitrogen species (RONS) in the PAW increased with longer plasma activation time. After being treated for 30 min in the PAW with a plasma activation time of 3 min, the spore viability dramatically dropped to 15%. The viabilities of 0.3% H2O2- and 0.3% HNO3-treated spores were 22% and 42%, respectively. The breakage of the spore cell wall by the PAW was revealed in scanning electron microscope images and flow cytometry measurements. Disruption of cell wall integrity provides a path for intracellular components to escape and RONS of the PAW can attack intracellular components directly. Degradation of high molecular genomic DNA was also observed by agarose gel electrophoresis. These results suggest that long-lived reactive species generated in the PAW play an important role in the inactivation of melanized fungal spores. Consequently, PAW produced by a soft plasma jet can be applied to sterilize bioprotective walled fungal spores in a relatively large volume.

Plasma-activated water (PAW) is enriched with reactive oxygen and nitrogen species (RONS), which significantly alter its physicochemical properties and expand its applicability in fields like materials science, biomedicine, and agriculture. This study investigates the specific contributions of key long-lived RONS—hydrogen peroxide (H2O2), nitrate ions (NO3 −), and ozone (O3)—to the physicochemical properties of PAW. PAW was produced using a pin-to-liquid plasma system, and its properties were characterized using UV–Vis spectroscopy, Raman spectroscopy, Fourier-transform infrared spectroscopy, and measurements of pH, electrical conductivity, total dissolved solids (TDS), and oxidation-reduction potential (ORP). To isolate the effects of individual RONS, aqueous solutions containing H2O2, NO3 −, O3, and a composite solution with the three species at concentrations equivalent to those measured in PAW were prepared and analyzed using the same characterization techniques. The individual RONS solutions revealed specific influences on the physicochemical parameters: H2O2 led to slight acidification and increased conductivity; NO3 −; significantly increased conductivity and TDS; and O3 had minimal effect on the measured properties. The composite solution is the only one that has a positive ORP; it is an oxidant. However, none of the individual solutions replicated the comprehensive alterations observed in PAW. The composite solution containing all three RONS showed more pronounced changes but still did not fully match PAW’s properties. This difference hints to the presence of unidentified long-lived reactive species in PAW, possibly originating from electrode degradation, as spectroscopic analyses indicate. Understanding the individual and combined effects of long-lived RONS is crucial for optimizing PAW generation and tailoring its properties for specific applications.

Plasma activated water (PAW) is formed during an exposure of water to the non-thermal plasma which induces some chemical changes in the treated water and produces reactive oxygen and nitrogen species (RONS) (e.g. hydrogen peroxide, nitrite, nitrate, peroxynitrite, hydroxyl radical, nitrogen monoxide radical, etc.). These RONS in PAW are responsible for the rapid germination of seeds, enhancement of the plant growth, destruction of bacteria and other germs harmful to the growth of the plants <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1, 2</sup> . PAW has also a role as a fertilizing agent for plants <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> .

In this paper, we employ UV absorption spectroscopy to monitor the generation and permeation of reactive oxygen and nitrogen species (RONS) in plasma-activated water (PAW) to revealthe dynamic variation mechanism of RONS chemistry. Parameters including gas impurity, pulse polarity and solution pH value are varied to explore their effects on the absorbance behavior and peak shift of absorption spectra as well as the permeation distribution of RONS. Regarding the absorbance behavior, experimental results show that introducing air and N2 into He working gas would effectively improve RONS absorbance, proportions of about 0.2% air and 0.5% N2 would result in the maximum absorbance, while the addition of O2 would result in a significant decrease in RONS absorbance. Under positive polarity, the RONS absorbance is about 20% higher than that under negative polarity. Changing the solution pH from acidic to alkaline is beneficial in increasing RONS absorbance, indicating that alkaline solution could effectively promote RONS formation. Regarding the characteristic peak shift, different parameter conditions seriously affect the shift of the absorption peak toward low wavelength or high wavelength due to the change in the ratio of the concentration of each component of RONS in PAW. Furthermore, with respect to the permeation distribution of H2O2 and NO2 −, the results show that the addition of O2 would result in the fastest production rate of H2O2 and introducing air and N2 would generate the fastest rate of NO2 − production. Interestingly, the NO2 − permeation distribution displays a ‘columnar mode’ and a ‘filamentous mode’ under positive and negative polarity, respectively. An alkaline solution promotes the formation of NO2 − while having an obvious inhibiting effect on the NO2 − permeation; conversely, an acidic solution has a promotional effect on NO2 −. This study provides a new in-depth understanding of the dynamic evolutionary behavior of RONS in PAW, helping to reveal the network relationship between RONS, and assisting in the development of applications of PAW.

Several attempts have been made to deliver reactive oxygen and nitrogen species (RONS) produced by non-thermal plasma onto large surfaces in a controllable way compatible with skin treatments. In this paper, the effect of pulse frequency, in the range 0.5–20 kHz, on the discharge behavior was studied on skin tissue models to evaluate potential treatment changes. This has been done through electrical characterization, visualization of the helium flow (by Schlieren technique), produced plasma jet modifications (ICCD imaging) and RONS measurements. The results show that, in addition to its well known important role in the production of the chemical species, the applied discharge frequency plays a very significant role in the size of the treated surface. An enhancement of NO*, OH* and O* production in the gas phase at the higher frequency is reported and assigned to the stronger mixing of the helium flow with ambient air. The efficacy of plasma jet on transporting RONS on/into agarose gel and pig skin has been evaluated. The distribution of the reactive species on the target, or passing through, is strongly dependent on the discharge frequency and consequently induces pH variations. The present study supports a new way for enlarging the treated surface by using a simple jet at high frequency in the 20 kHz range, leading, with appropriate gas flow and distance to target, to conditions of RONS production that are compatible with potential uses for biomedical or cosmetic applications.

Cold atmospheric pressure plasma jets (plasma) operated in ambient air provide a rich source of reactive oxygen and nitrogen species (RONS), which are known to influence biological processes important in disease. In the plasma treatment of diseased tissue such as subcutaneous cancer tumors, plasma RONS need to first traverse an interface between the plasma-skin surface and second be transported to millimeter depths in order to reach deep-seated diseased cells. However, the mechanisms in the plasma generation of RONS within soft tissues are not understood. In this study, we track the plasma jet delivery of RONS into a tissue model target and we delineate two processes: through target delivery of RONS generated (primarily) in the plasma jet and in situ RONS generation by UV photolysis within the target. We demonstrate that UV photolysis promotes the rapid generation of RONS in the tissue model target's surface after which the RONS are transported to millimeter depths via a slower molecular process. Our results imply that the flux of UV photons from plasma jets is important for delivering RONS through seemingly impenetrable barriers such as skin. The findings have implications not only in treatments of living tissues but also in the functionalization of soft hydrated biomaterials such as hydrogels and extracellular matrix derived tissue scaffolds.

Cold plasma generated by atmospheric pressure air discharge is a source of various gaseous reactive oxygen and nitrogen species (RONS). When the plasma is generated in a contact with water, the RONS dissolve into water, change its chemical composition, while producing so-called plasma activated water (PAW). The PAW has the potential to be effectively used in various agricultural applications, as the long lived liquid RONS (H2O2, NO2−, NO3−) may act like signaling molecules in plant metabolism or serve as nutrients. We studied the effect of the PAW on lettuce plants and compared it with the effect of H2O2 and/or NO3− solutions of various concentrations to assess their role in the PAW. The PAW was generated from tap water by DC driven self-pulsing transient spark discharge. Pre-grown lettuce plants were cultivated in pots with soil and irrigated with the PAW or solutions of H2O2 and/or NO3−. After 5 weeks the growth parameters, number and quality of leaves, fresh and dry weight of plants, photosynthetic pigment (chlorophyll a + b) content, photosynthetic rate, and activity of antioxidant enzymes (superoxide dismutase, SOD) were evaluated. Lettuce plants irrigated with the PAW in comparison with chemically equivalent solution of H2O2 and NO3− had similar dry weight; however, the PAW induced higher photosynthetic pigment content, higher photosynthetic rate, and lower activity of SOD. The NO3− mainly contributed to the increase of dry weight, photosynthetic pigment content, photosynthetic rate, and overall better appearance of plants. The H2O2 contributed to an increase of dry weight and induced SOD activity. In general, H2O2 and NO3− in proper concentrations can stimulate plant growth and affect their physiological properties.

Due to their potential benefits, cold atmospheric plasmas (CAPs), as biotechnological tools, have been used for various purposes, especially in medical and agricultural applications. The main effect of CAP is associated with reactive oxygen and nitrogen species (RONS). In order to deliver these RONS to the target, direct or indirect treatment approaches have been employed. The indirect method is put into practice via plasma-activated water (PAW). Despite many studies being available in the field, the permeation mechanisms of RONS into water at the molecular level still remain elusive. Here, we performed molecular dynamics simulations to study the permeation of RONS from vacuum into the water interface and bulk. The calculated free energy profiles unravel the most favourable accumulation positions of RONS. Our results, therefore, provide fundamental insights into PAW and RONS chemistry to increase the efficiency of PAW in biological applications.

Low-temperature plasma (LTP) has demonstrated great potential in biomedicine, especially in cancer therapy in-vivo and in-vitro. Plasma activated water (PAW) as an indirect plasma therapy is a significant source of reactive oxygen and nitrogen species (RONS) which play an important role in apoptosis induction in cancer cells. In this study, Helium (He) plasma jet operating in 0.75 W and 20 kHz as dissipated power and frequency, respectively, is used as the cold plasma source. The electrical, thermal, and spectroscopic properties of (He) plasma jet and pH as well as the conductivity and temperature of PAW samples, are investigated. The concentration of hydrogen peroxide (H2O2), nitrite (NO2-) and nitrate (NO-3), which are produced in water as long-lived anticancer RONS, was measured 471.6, 7.9 and 93.5 μM, respectively after 6 min of plasma treatment. Alamar Blue and flow cytometry assays were employed to investigate the B16F10 cancer metabolic activity and apoptosis. These data support that cold atmospheric plasma (CAP) can produce a certain concentration of anti-cancer agents in water and induce apoptosis in melanoma cancer cells due to RONSs via activating the caspase 3 pathway.

The aim of this study is to investigate the impact of long-lived and short-lived reactive oxygen and nitrogen species (RONS) generated by cold atmospheric plasma on protein. In contrast to existing research primarily focusing on the effects of RONS on proteins, this study emphasizes the clarification of the effects of long-lived and short-lived RONS on proteins and potential subsequent impacts on cells. Through both direct and indirect plasma treatment, we compared the effects of long-lived and short-lived RONS on proteins and observed protein structural changes using techniques such as gel electrophoresis and Western blotting. The results indicate that peptide bonds and intramolecular disulfide bonds in proteins remain intact, but the binding affinity of TNF-&amp;alpha; with antigens decreases. Since both methods of application resulted in decreased binding affinity, it is inferred that long-lived RONS are the primary cause. In summary, the RONS generated by the plasma jet used in this study (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; concentration of 867 &amp;mu;M, &amp;#183;OH concentration of 60 &amp;mu;M) did not alter protein structures. Furthermore, when compared to other studies on cancer cell apoptosis, the RONS concentration in this study was higher, but protein structure remained unchanged. Therefore, it is suggested that RONS do not induce cell apoptosis through the direct disruption of protein structures.