Sticking out needle from perspex tube has less effect than increasing distance

1. position distance Linear time dependence Distance: less than 1/r2 ! Position: less important Extra oxygen, no radical increase Use of a fluorescent probe to detect radicals formed by the plasma needle Ingrid E. Kieft, Joep J.B.N van Berkel, Debbie Bronneberg, Eva Stoffels, Dick W. Slaaf Department of Biomedical Engineering, Eindhoven University of Technology P.O.Box 513, 5600 MB Eindhoven, The Netherlands E-mail: I.E.Kieft@tue.nl Introduction Study of interaction of small atmospheric discharge with living cells [1,2] Use of Plasma Needle [3] Effect on cell culture: cell detachment Results and discussion Fluorescence measurements of radical concentration Cell detachment in time. 1) control sample, 2) 15 min. after treatment, 3) 1 hour after treatment, 4) 4 hours after treatment. Calibration of fluorescence by NOR-1 From 10 µM curve flattens - saturation Effect on tissue engineered skin: First test shows formation of vacuoles and detachment Cross section of tissue engineered skin. Control sample (left) and sample treated for 5 min (right). Radical concentration in µM range – fysiological concentration Distance of tip needle-surface liquid > 2mm radical concentration less than half Plasma applied  cells detach and remain alive What causes this cell reaction? Is it caused by radicals? Detect and measure radicals Material and methods CM-H2DCFDA probe: is dissolved in liquid becomes fluorescent upon reaction with radical Probe reacts to: H2O2, HO., HOO., ONOO- and NO • Sticking out needle from perspex tube has less effect than increasing distance • Adding more oxygen less radicals • 1 % O2  factor 10 decrease • Conclusions • Cells detach after plasma treatment, and remain alive. They are capable of reattachment and cytokinesis. • Fluorescent probe is a good and easy to use method to detect radicals in liquids • Radicals reach the liquid in fysiological concentrations • More molecules in plasma leads to less radicals, due to loss of electrons and energy. • The high precision plasma treatment can have applications in wound healing and cancer treatment. Fluorescent probe before and after activation and reaction with radical In situ picture  Calibration with NO releaser NOR-1 Releases NO within 15 min after dissolving Measurements performed with microplatereader • Plasma parameters • Plasma power is 50 to 100 mW • Voltage peak to peak 200 - 400 V • Helium flow 2 l/min • Irradiation time of sample is 2 minutes • Irradiated volume 400 µl References: [1] Stoffels,E., Kieft, I.E., Sladek, R.E.J., Superficial treatment of mammalian cells using plasma needle, J.Phys.D: Appl. Phys. 36 (2003) 2908-2913 [2] Kieft, I.E., Broers, J.L.V., Caubet-Hilloutou, V., Slaaf, D.W., Ramaekers, F.C.S., Stoffels, E., Electric discharge plasmas influence attachment of cultured CHO K1 cells, accepted for publication in Bioelectromagnetics [3] Stoffels, E., Flikweert, A.J., Stoffels, W.W., Kroesen, G.M.W., 2002, Plasma Sources Sci. Technol. 11: 383-388