Magesh Thiyagarajan

Characterization of Portable Resistive Barrier Plasma Jet and Its Direct and Indirect Treatment for Antibiotic Resistant Bacteria and THP-1 Leukemia Cancer Cells

An atmospheric pressure nonthermal resistive barrier plasma (RBP) jet was designed, constructed, and characterized for plasma surface treatment procedures applied in biomedical applications. The RBP source can operate in both dc (battery) and standard 60/50-Hz low-frequency ac excitation and can function effectively in both direct and indirect plasma exposure configurations, depending on the type of treatment targets and applications. The design and construction aspects of the RBP source are presented, including the electrode configuration, electrical, cooling, and gas flow aspects. The RBP jet is tested, and its characteristics such as the propagation velocity of the plasma jet, electrical properties, plasma gas temperature, and nitric oxide concentration are characterized using optical laser plasma shadowgraphy, voltage-current characterization, optical emission spectroscopy (OES), and gas analyzer diagnostic measurements, respectively. Using a laser shadowgraphy diagnostic, we have measured the average propagation velocity of the plasma jet to be 150-200 m/s at 1 cm from the probe end. The discharge power is calculated from voltage-current characterization, and the plasma power is 26.33 W. An OES was applied, and the gas temperature which is equivalent to the nitrogen rotational (Trot) temperatures was measured. After approximately 2 cm from the tip, along the axis, the plasma emission drops, and the high-temperature ceramic fiber-insulated-wire thermocouple probe was used to measure the temperatures of the gas flows along the downstream jet. The addition of a small portable external cooling unit has brought the temperatures of reactive oxygen species (ROS) and other gases close to room temperature at the tip of the handheld plasma source unit. The concentrations of the ROS at different spatial distances from the tip of the plasma jet were measured; at a 5-cm distance from the electrode, the nitric oxide level was measured to be in the range of 500-660 ppm, and it drops to ~ 100 ppm at 60 cm. The parts-per-million values of nitric oxides after the cooling unit are observed to be of the same order of magnitude as compared to that of the plasma jet. The portable RBP source was tested to be very effective for the decontamination and disinfection of a wide range of food-borne and opportunistic nosocomial pathogens such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus cereus, and the preliminary results are presented. The effects of the indirect exposure of the portable RBP source on monocytic leukemia cancer cells (THP-1) were also tested, and the results demonstrate a preference for apoptosis in plasma-treated THP-1 cells under particular plasma parameters and dosage levels.

Induction of apoptosis in human myeloid leukemia cells by remote exposure of resistive barrier cold plasma

Cold atmospheric plasma (CAP), an ambient temperature ionized gas, is gaining extensive interest as a promising addition to anti-tumor therapy primarily due to the ability to generate and control delivery of electrons, ions, excited molecules, UV photons, and reactive species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) to a specific site. The heterogeneous composition of CAP offers the opportunity to mediate several signaling pathways that regulate tumor cells. Consequently, the array of CAP generated products has limited the identification of the mechanisms of action on tumor cells. The aim of this work is to assess the cell death response of human myeloid leukemia cells by remote exposure to CAP generated RNS by utilizing a novel resistive barrier discharge system that primarily produces RNS. The effect of variable treatments of CAP generated RNS was tested in THP-1 cell (human monocytic leukemia cell line), a model for hematological malignancy. The number of viable cells was evaluated with erythrosine-B staining, while apoptosis and necrosis was assessed by endonuclease cleavage observed by agarose gel electrophoresis and detection of cells with the exclusionary dye propidium iodide and fluorescently labeled annexin-V by flow cytometry and fluorescent microscopy. Our observations indicate that treatment dosage levels of 45□s of exposure to CAP emitted RNS-induced apoptotic cell death and for higher dosage conditions of ≥ 50□s of exposure to CAP induced necrosis. Overall the results suggest that CAP emitted RNS play a significant role in the anti-tumor potential of CAP.

Experimental Investigation of 1064-nm IR Laser-Induced Air Plasma Using Optical Laser Shadowgraphy Diagnostics

Experimental measurements and analysis of pulsed 1064-nm Nd:YAG laser-induced air breakdown plasma at 760 torr has been carried out using high-speed and high-resolution laser shadowgraphy and optical diagnostics. Three different experimental laser energies and pulsewidths such as 170 mJ at 8 ns, 130 mJ at 7 ns, and 65 mJ at 12 ns are studied. The laser pulses were focused down to a ~ 7-μm spot size in air and the resulting laser flux densities range from 4 to 14 TW/cm2. A 532-nm laser shadowgraphy coupled with high-speed and high-resolution image capturing diagnostics has been established to investigate spatiotemporal evolution and hydrodynamic behavior of the 1064-nm laser-induced plasma and neutral-density shock during the formation, expansion, and collapsing stages. The observed plasma formations were aspherical due to absorption translation during the initial laser-energy coupling. The aspherical feature seeded the hydrodynamic instability leading to the ultimate destabilization of the hot gaseous core after approximately 10 μs. The active plasma lifetime through plasma self-luminescence measurements indicate variations from 200 to 500 ns for the three laser pulses. Shock propagation velocity and plasma volume for three laser pulse series indicate similarly shaped profiles at different expansion velocities. Early plasma expansion velocities of 20 km/s were measured, and using Hugoniot relations, the neutral shock pressures and temperatures were inferred, and the results at the early plasma expansion stage were found to be over 1000 atm and 4 eV.

Portable plasma torch treatment on E. coli, S. aureus, N. meningitidis and other clinical isolates

The uses of non-thermal plasmas in medical, environmental and consumer industries especially in food processing, air filtration, decontamination and sterilization of bacteria in liquids and solids surfaces, are continued to grow. In this paper we present the research efforts on the effects of a portable non-thermal plasma source treatment on bacteria in liquid phases. We predict that the plasma species can penetrate/diffuse in to the liquid culture media and plasma treatment will efficiently kill the bacteria at unique time and distance parameters. It is also hypothesized that less stringent plasma treatment will negatively affect the growth rate of some species of bacteria and possibly their pathogenicity. Cultures used were at optical densities (O.D.600nm) ranging from 0.2 to 1.0 or McFarland standards of 0.5 and higher. The bacteria were exposed to plasmas at various time lengths and distance parameters and gas temperatures. Our results indicates that less than 120 seconds of plasma treatment with the plasma gun ∼5 cm away from the liquid culture is effective in killing/sterilizing cultures of S. aureus, S. pyogenes, Salmonella spp, N. meningitidis, and E. coli. Less than five minutes of cold plasma with the probe immersed 1–2 cm inside the liquid culture were needed to kill the bacteria. Furthermore, growth curve analysis showed that N. meningitidis, S. aureus and E. coli exposed to less than 2 minutes of treatment experienced decreased growth. N. meningitidis exposed to 1 minute of hot plasma treatment displayed a heavier mucoid appearance on agar plates and subsequent capsule staining will be employed to verify microscopically if a larger capsule is present in comparison to untreated samples. Furthermore, q-real time-RT-PCR will be employed with N. meningitidis to determine quantitative amount of capsule gene expression using primers for synC and synD genes (needed for capsular polysaccharide synthesis) and the ctrA gene (needed for capsular polysaccharide transport). Additionally, q-real time-RT-PCR work will be used to investigate whether N. meningitidis has decreased synthesis of the PilC gene after 1 minute treatment with hotter plasma. The PilC gene codes for the bacterias pili, which are used for adhesion and pathogenicity in its host. Subsequent studies should be done to explore the effects of plasma treatment on the pathogenicity of different species of bacteria.

Atmospheric pressure resistive barrier low temperature plasma treatment for food industry

Summary form only given. An atmospheric pressure resistive barrier air plasma jet is designed to inactivate bacteria in food media such as chicken, egg, seafood, vegetables and fruits1,2. Resistive barrier plasma is designed to function at both DC and standard 50-60 Hz low frequency AC power input and the ambient air at 50% humidity level was used as the operating gas. The resistive barrier plasma is well characterized1. The nitric oxides (NO) were observed to be the predominant long lived reactive nitrogen species produced by the plasma. Three different bacteria including S. aureus, E. coli and N. meningitidis were tested in aqueous media and positive bacterial inactivation results were obtained with plasma treatment. Similarly, three different bacteria including S. aureus, E. coli and Pseudomonas were tested on dry solid surfaces and positive bacterial inactivation results were obtained in less than 180 seconds. Correspondingly, the frequent food poisoning bacteria Salmonella infected poultry and produce such as chicken, eggs, vegetables and fruits were tested using our atmospheric pressure resistive barrier air plasma jet and the results show significant outcomes in terms of bacterial inactivation. Plasma induced shifts in gene expression were analyzed using pilC gene expression as a representative gene and the results showed a reduction in the expression of the pilC gene compared to untreated samples suggesting that the observed protection against NO may be regulated by other genes.

Scalable nanoparticle synthesis in liquids using laser induced plasmas at phase boundaries

Summary form only given. Nanofluids are a new class of fluids engineered by dispersing nanoparticles of size less than 100 nm in base fluids. Nanofluids have been found to possess highly enhanced physical, chemical, thermal and transport properties compared to the base fluids, which demonstrates the great potential for combustion, liquid propellant, microelectronics, optical and thermal emission devices, energy storage, heat exchanger-cooling systems, hydrogen generation, nuclear safety, and in underwater and military applications. In this experimental investigation the feasibility results of producing scalable enhanced nanoparticles in liquids laser induced plasmas at liquid-metal (Al) phase boundaries using our laser plasma facility will be presented. The formation and dynamics of laser plasmas and shock waves at liquid-metal phase boundary was affected by the conditions of strong liquid confinement. The plasma and shock spatio-temporal dynamics and velocities varied for different laser transfer matrix and experimental conditions. The plasma electron density of the laser induced plasma at liquid-Al phase boundaries was measured using a two-wavelength laser interferometry. In order to better understand the relationship and synthesis of effective nanofluids the preliminary results of correlating the plasma characteristics with the nanoparticles size and size distribution will be presented.

Laser shadowgraphy, two-wavelength laser interferometry, schlieren imaging and optical emission spectroscopy diagnostics of laser induced plasmas in different phases and at phase boundaries

Summary form only given. Fast gating and high resolution laser shadowgraphy, two wavelength laser interferometry, Schlieren imaging and optical emission spectroscopy diagnostics were carried out to experimentally investigate pulsed 1064 nm Nd:YAG laser-induced breakdown plasma in air at 760 Torr1 and in liquid (water) and in liquid-metal phase boundaries. Three different experimental laser energies and pulse widths such as 170 mJ at 8 ns, 130 mJ at 7 ns and 65 mJ at 12 ns are studied. The laser pulses were focused down to a ~7 micron spot size in air and the resulting laser flux densities range from 4-14 TW/cm2. The detailed experimental arrangements and results will be presented. A 532 nm laser shadowgraphy coupled with high speed and high resolution image capturing diagnostics has been established to investigate spatiotemporal evolution and hydrodynamic behavior of the 1064 nm laser induced plasma and neutral density shock during the formation, expansion and collapsing stages. The active plasma lifetime through plasma self-luminescence measurements indicate variations from 200-500 ns for the three laser pulses. Shock propagation velocity and plasma volume for three laser pulse series indicate similarly shaped profiles at different expansion velocities. Early plasma expansion velocities of 20 km/s were measured and using Hugoniot relations the neutral shock pressures and temperatures were inferred and the results at the early plasma expansion stage were found to be over 1000 atmospheres and 4 eV. Laser induced plasma breakdown and its resulting shockwave generation and bubble formation in water was also investigated and characterized. The results of these investigations will be presented. Two wavelength laser interferometry based plasma density measurements will be presented. Plasma temperature measurements using optical emission spectroscopy diagnostics will be presented.

Characterization of non-thermal atmospheric pressure helium plasma jet for biomedical applications

In this work, an atmospheric pressure plasma jet generated in a dielectric barrier discharge (DBD) configuration with He and He/O2 mixture has been characterized using electrical and optical emission spectroscopy (OES) diagnostics. A visible plasma plume length of approx. 33 mm was generated using a 60 kHz AC power supply. The discharge characteristics in the active and afterglow region of the plasma jet that are critical for biomedical applications have been investigated. Atomic and molecular lines of selected reactive plasma species that are considered to be u seful to induce biochemical reactions in biomedical applications such as OH transition at 308 nm and OH transition at 287 nm, O I transition 3p5P→3s5S0 at 777.41 nm, O I transition 3p3P→3s3S0 at 844.6 nm, N2(C-B) second positive system (SPS) with electronic transition in the range of 300-450 nm and N2+(B-X) first negative system (FNS) with electronic transition at 391.4 nm have been studied. The OH rotational temperatures that are equal to gas temperatures was measured based on the fitting of experimental spectra on to simulated spectra of the plasma jet. Our results show that, in the entire active plasma region, the gas temperature remains at 380 K and it increases to 400 K in the afterglow region of the plasma jet. In conclusion, the plasma jet produces significant reactive species while gas temperature remains cold and therefore it can be applied to a range of biomedical applications12.

Experimental investigation of 1064 nm IR laser induced plasmas in gases and in liquids

Experimental measurements and analysis of pulsed 1064 nm Nd:YAG laser-induced breakdown plasma in air at 760 Torr and in liquid (water) has been carried out and characterized using fast gating and high resolution laser shadowgraphy, two wavelength laser interferometry and optical emission spectroscopy diagnostics1. Three different experimental laser energies and pulse widths such as 170 mJ at 8 ns, 130 mJ at 7 ns and 65 mJ at 12 ns are studied. The laser pulses were focused down to a ~7 micron spot size in air and the resulting laser flux densities range from 4-14 TW/cm2. A 532 nm laser shadowgraphy coupled with high speed and high resolution image capturing diagnostics has been established to investigate spatio-temporal evolution and hydrodynamic behavior of the 1064 nm laser induced plasma and neutral density shock during the formation, expansion and collapsing stages. The observed plasma formations were aspherical due to absorption translation during the initial laser-energy coupling. The aspherical feature seeded the hydrodynamic instability leading to the ultimate destabilization of the hot gaseous core after approximately 10 microseconds. The active plasma lifetime through plasma self-luminescence measurements indicate variations from 200-500 ns for the three laser pulses. Shock propagation velocity and plasma volume for three laser pulse series indicate similarly shaped profiles at different expansion velocities. Early plasma expansion velocities of 20 km/s were measured and using Hugoniot relations the neutral shock pressures and temperatures were inferred and the results at the early plasma expansion stage were found to be over 1000 atmospheres and 4 eV. Laser induced plasma breakdown and its resulting shockwave generation and bubble formation in water was also investigated and characterized. The results of these investigations will be presented.

Activation of apoptotic cell death in human myeloid leukemia cells by RNS: A novel antitumor approach using resistive barrier plasma

Cold atmospheric plasma (CAP), an ambient temperature ionized gas, is undergoing extensive evaluation as a promising addition to anti-tumor therapy primarily due to the ability to generate and control delivery of electrons, ions, free radicals, excited atoms and molecules, UV photons, and reactive species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) to a specific site. The heterogeneous composition of CAP offers the opportunity to mediate several signaling pathways that regulate tumor cells. Consequently, the array of CAP generated products has limited the identification of the mechanisms of action on tumor cells. The aim of this work was to assess the cell death response of human myeloid leukemia cells exposed to CAP generated RNS. We evaluated the viability of the human monocytic leukemia cell line THP-1 to variable treatments of CAP generated RNS and we set to identify specific cell death characteristics that presented with increasing dosages of CAP generated RNS. Incorporation of exclusionary dyes and fluorescently labeled annexin V allowed us to determine that CAP generated RNS promotes apoptosis and/or necrosis in THP-1 cells in a dosage- and time-dependent manner. CAP generated RNS promotion of apoptosis was further supported by observation of endonuclease cleavage products by agarose gel electrophoresis. In conclusion, we identify that CAP generated RNS play a significant role in the anti-tumor potential of CAP and show a possible application of modified CAP devices in tumor therapy1, 2. Recent results on RNA analysis will be presented as well.