| Due to the characteristics of low temperature,high efficiency and environment-friendly,cold atmospheric pressure plasmas?CAPPs?has attracted more and more attentions.Researchers have found that it has a promising application prospects,such as sterilization,cancer treatment,food safety,environmental protection.Undoubtedly,CAPPS could efficiently kill various microorganisms,the difference of plasma generator devices,numerous components in plasma as well as complexity of biological responses,the mechanism of CAPPS sterilization against microorganisms needs to be further explored.Surface micro-discharge?SMD?plasma with a large-area and homogeneous discharge has attracted much attention in the skin disinfection due to the high antimicrobial efficiency and less side-effects on tissues.Although SMD plasma sterilization is undisputedly attributed to the reactive oxygen and nitrogen species?RONS?,the exact RONS speciation and their individual contribution to the plasma inactivation are still not fully understood.Therefore,in this thesis,RONS speciation in gas,tissue and liquid phase was investigated.In addition,Saccharomyces cerevisiae?S.cerevisiae?was used as the experimental object,the mechanism of SMD plasma induced S.cerevisiae inactivation was detailed studied.The research work presented here can be categorized into three parts:?1?Comparative study the major antimicrobial agents on the tissue model by helium and air surface micro-discharge plasma:We investigated the generation and distribution of hydroxyl radical?·OH?,hydrogen peroxide?H2O2?,ozone?O3?,nitrite?NO2??and peroxynitrite/peroxynitrous acid?OONO?/ONOOH?on the agrose tissue model and their contribution to yeast inactivation by helium?He?or air SMD plasma at different irradiation distance.The results show that He and air SMD plasma exhibited different RONS speciation and antimicrobial activity.He SMD plasma mostly generated·OH and H2O2on the tissue model,which were concentrated in every hexagon micro-discharge unit and decreased with the irradiation distance.Whilst,air SMD plasma mainly produced O3,NO2?and OONO?/ONOOH,which were uniformly distributed on the whole tissue model.More importantly,the·OH generation on the tissue model by He SMD plasma was derived from the plasma delivery,while UV photolysis led to the situ·OH generation by air SMD plasma.Additionally,the air SMD plasma has a higher inactivation efficiency than He SMD plasma and the major antimicrobial agent for He and air SMD plasma is respectively·OH and O3in this plasma-tissue interaction system.?2?Evaluating the roles of OH radicals,H2O2,ORP and p H in the inactivation of yeast cells on a tissue model by surface micro-discharge plasma:In this part,we further investigate the inactivation effect of helium SMD plasma on yeast in the irradiation distance of 1,2 and 3 mm.·OH and H2O2generation and distribution on tissue model surface were investigated.We also examined the electrochemical parameters in the tissue surface micro-environment.Pearson correlation analysis demonstrated that·OH plays a major role in killing yeast cells,which is consistent with the results of the first part of the study.Furthermore,we investigated the effects of·OH and p H on yeast cell viability,membrane integrity and intracellular ROS and p H homeostasis by using a specific·OH scavenger D-mannitol?D-man?and phosphate buffer solution?PBS?.The results showed that D-man could significantly reduce the inactivation efficiency by maintaining cell membrane integrity and intracellular ROS and p H homeostasis,while PBS only slightly mitigates the plasma-caused damage on yeast cells.Based on the results,it is concluded that·OH contributes most to the inactivation of yeast cells on a tissue model by helium SMD plasma studied here.?3?The study of yeast inactivation mechanism induced by ROS generated in the SMD plasma-liquid interaction system:In this study,subcellular mechanism of yeast cell inactivation during plasma-liquid interaction was revealed in terms of comprehensive factors including cell morphology,membrane permeability,lipid peroxidation,membrane potential,intracellular redox homeostasis?intracellular ROS and H2O2,SOD,CAT,GSH?,intracellular ionic equilibrium?intracellular H+and K+?and energy metabolism(mitochondria membrane permeability,intracellular Ca2+,cellular ATP level).The ROS analysis show that·OH,1O2,O2-and H2O2were generated in this plasma-liquid interaction system.In addition,the solution p H was reduced.ROS scavengers?L-His,D-Man and SOD?and p H buffer?phosphate buffer solution,PBS?were employed.It was found that every antimicrobial factors?·OH,1O2,O2-,H2O2or low p H?have different influences on the aforementioned cell physiology.Results demonstrated that·OH and 1O2contributes the most to the yeast inactivation.·OH could attack cell membrane and increase membrane permeability.O2-served as the precursor of 1O2and the changes of cell energy metabolism was mainly attributed to damage of 1O2.During the plasma inactivation process,yeast cells mainly underwent apoptosis in the first 5 min mainly due to the destroy of intracellular antioxidant system by intracellular ROS?·OH and1O2?and the disorder of cell energy metabolism.In addition,the damage of cell membrane as well as extracellular low p H could break the intracellular ionic equilibrium and further reduce cell membrane potential.PBS could maintain the stable of intracellular ions and postpone the cells death.10-min treatment could cause necrosis of yeast cells.The remarkable increase of intracellular H2O2play a little role in inactivating yeast cells.The above results reveal the major reactive species produced by surface micro-discharge plasma in tissue and liquid phase and its antimicrobial mechanism against S.cerevisiae,which is of great significance for the application of surface micro-discharge plasma in the field of skin and water sterilization. |
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