Penicillium digitatum spores interacted with plasmas

Transcription

1 JSAP 2011 Fall 31a-ZD-10 Electron-spin-resonance (ESR) study of Penicillium digitatum spores interacted with plasmas Kenji Ishikawa, Sachiko Iseki, Takayuki Ohta*, Masafumi Ito*, Hiroshi Hashizume*, Hiroyuki Kano**, Keiko Takeda, Hiroki Kondo, Makoto Sekine, and Masaru Hori Plasma Nanotechnology Research Center (PLANT) Nagoya University, *Meijo University, **NU-eco 1/13

2 Background Outline Fungal spore of Penicillium digitatum Inactivation mechanism Plasma and biomaterial interaction Experimental Free radical electron spin resonance (ESR) Results and discussion ESR signal identification Semiquinone? Individual contribution of radical and light Real time measurement during plasma irradiation Summary 2/13

3 Fugal spores of Penicillium digitatum inactivation Atmospheric pressure plasmas of trace oxygen containing argon gas. No contribution of UV radiation, NO, and O 3. Effect of O atom or OH Postharvest disease, cause green mold of citrus fruits survive UV Radical +UV inactivate after Iseki, APL 96, (2010). Q: Inactivation mechanism? 3/13

4 Inactivation mechanism Biological responses for disinfections by the plasma discharges 1. Chemical - lethal oxidation of membranes, 2. Physical - Disruption of cell membranes, cell lysis, fragmentation 3. Biological - intracellular signaling, apoptosis, 4. etc. polysaccharide (sugar) mitochondrion, etc (energy metabolism) Intracellular auto-oxidation Free radical Nuclei, DNA after Imlay, Ann Rev Microbiol (2003). 4/13

5 Radical detection in situ at real time H Real time in situ electron spin resonance (ESR) Energy, E 0 0 Dangling bond h ν (9 GHz) S= - 1/2 g β H S= + 1/2 Electron spin (S = ±½) ca. 300mT Magnetic field, H g : g-value (free electron ) β : Bohr magnetron Dangling bond formation on fluoropolymer (PTFE) interacted with Hydrogen plasma Radical formation speed 4 2 Radical + Light Light Radical Time [min] after Ishikawa, J. Phys. Chem. Lett. (2011). 5/13

6 Purpose of this study Elucidation of inactivation mechanisms on living cells and tissues on bacteria by exposing to plasmas Approaches Fungal spore of Penicillium digitatum Electron-spin-resonance (ESR) detection and identification of free radical on the bacteria Real time in situ measurements during reaction under plasma exposure 6/13

7 Sample preparation Sample: Fungal spore of Penicillium digitatum 1. Dispersion in distilled water with Tween20 avoiding clump of spores 2. Coating on glass tube of sample (immerse and dry) ESR tube (1.6mm dia.) 1 2 ESR tube (5mm dia.) Sample with water 7/13

8 ESR spectra of Penicillium digitatum three Gaussian signals located at g=2.0030, 2.5 G, g=2.0040, 5.2 G, g=2.0014, 2.5 G. Spore of Penicillium digitatum has free radical at sporulation phase Note: after germination of sprout, no ESR signal was detected. Decolorization Note: for reagent of D-glucose, no ESR signal detected. 8/13

9 ESR signal assignment semiquinone? similar signal observed other experiments. g=2.0035, 5G NADPH FADH Molecules on (energy) metabolism naphthoquinone, after Pryor, Science (1983). after Kalyanaraman, Biochim. Biophys. Acta., (1980). anthracycline semiquinone 9/13

10 Real time ESR apparatus All contributions (Atomic O and Light) Plasma source ESR cavity Gas inlet : Neutrals : Light Exhaust Microwave Power (2.45 GHz) d Quartz tube O.D. 5mm with Fungal spore Radical (Atomic O) Light shade Quartz tube I. D. 10 mm He or O 2 Low pressure plasma 10 Pa 10/13

11 with U-shaped tube : atomic O contribution ESR intensity (arb. unit) spore atomic O exposure O 2 gas 240s 180s 120s 60s Gas ESR signal in spore decreased by exposure of atomic oxygen It might be oxidized the semiquinone as stable radical inside fungal spores. O* nuclei Initial g-value Reductase* R* + O -> R-O 11/13

12 Real-time measurement: ESR signal decay spore 3.5 initial level ESR intensity (arb. unit) O plasma exposure Integrated ESR intensity (arb. unit) Exposure time (min) Decay rate was a constant -> Oxidant transport-limited? g-value /13

13 Summary We have measured the ESR signal arisen from the fungal spores of Penicillium digitatum as an example of living bacteria. The ESR signal was decreased by only exposure of atomic oxygen (O*). It is believed that the ESR signal is assigned to semiquinone inside the spore, and was oxidized by introduction of the atomic oxygen. Anyway the in situ real time ESR method opens a new approach of analysis about interactions between plasma and biomaterial surface. 13/13

14 Thank you for your kind attention Related article: Appl. Phys. Lett. 96, (2010), J. Phys. Chem. Lett. 2, 1278 (2011). Acknowledgement This was partly supported by Tokai region of the Knowledge Cluster Initiative (The Second Stage). Author: Kenji Ishikawa 14/13