Plasma-Liquid Chemistry in the Presence of Organic Matter - Katharina Stapelmann Assistant Professor
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Plasma-Liquid Chemistry in the
Presence of Organic Matter
Katharina Stapelmann
Assistant Professor
Department of Nuclear Engineering, North Carolina State University
DE-SC0021329
Overview
§ Plasma-Liquid Chemistry for Bio Applications
§ Organic matter in liquids becomes part of the chemistry
§ “Passive”: offering a target for reactions
§ “Active”: cells actively change chemistry
https://doi.org/10.1088/0963-0252/25/5/053002 2
Motivation
§ Plasma Medicine: cell culture treatment
§ Cell medium different reactions than in water Plasma 2018, 1(1), 47-60
§ How do cells impact the liquid chemistry?
§ Plasma Agriculture: treatment of water
§ How clean is the water?
§ Transition to real-life applications
How does the chemistry change when organic www.plasmaleap.com
matter is present? 3
COST Reference Microplasma Jet
§ Designed as reference source:
robust and reproducible
§ Allows to contextualize results
§ CCP, 13.56 MHz RF, He, He/H2O
and He/O2
§ 1 mm electrode distance, 30 mm
plasma channel
§ Integrated matching network along
with current and voltage probes for
COST Reference Microplasma Jet continuous monitoring
4
Golda J et al. Journal of Physics D: Applied Physics. 2016;49(8):84003.
Experimental Conditions
§ Total gas flow: 1 slm
§ Power constant at 750 mW
§ ~240 VRMS for He/O2 and He/H2O
§ ~215 VRMS for He
§ Liquid treatments:
§ 12-well plate, 1 ml treatment volume
§ 4 mm distance from nozzle to liquid
§ All measurements performed in triplicate
5
He 0.6% O2 He-only He 2500 ppm H2O
O O ●OH ●OH OH-
O2●-
O3 He*
1O HO2● H2O2
2
6
O and OH
in He/O2 and He/H2O
He 1% O2 He 2500 ppm H2O
1013 1015 1014
7.5 0 3 0 2
Atomic oxygen density / cm-3
2 2
1.5
5 2
4 4
1
6 6
2.5 1
0.5
8 8
0 10 0 10 0
-1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1
Position / mm Position / mm
§ O (TALIF) and OH (LIF) in an open effluent
and with a liquid interface present at 4 mm
distance
§ O dominates in He/O2, OH in He/H2O
Myers, B. et al. Journal of Physics D: Applied Physics 54 (2021): 145202 7
Stapelmann, K. et al. Journal of Physics D: Applied Physics 54 (2021): 434003
Start simple: H2O2 formation in DI water
§ H2O2 in He/H2O plasma-treated
sample is ~30x higher than He,
x50 ~50x higher than He/O2
x30 § Corresponds well to OES and
previously reported gas phase
measurements*
*Benedikt J et al. Plasma Sources Sci
Technol. 2016;25(4):045013. 8
Isolating H2O2 origins using OH scavenger
H2O2 is produced primarily in gas phase H2O2 is produced primarily in aqueous phase
●OH ●OH ●OH ●OH
H2O2 H2O2 H2O2 H2O2
●OH ●OH ●OH ●OH
H2O2 H2O2 H2O2 H2O2
H2O2 ●OH ●OH DMPO–OH H2O2
H2O2 H2O2 H2O2 H2O2 H2O2 H2O2
DMPO DMPO–OH
H2O2 H2O2 DMPO DMPO H2O2 ●OH H2O2 H2O2
H2O2 ●OH DMPO–OH
DI water 50 mM DMPO DI water 50 mM DMPO 9
Isolating H2O2 origins using OH scavenger
§ OH scavengers: Terephtalic acid
(TA) and spin-trap 5,5-Dimethyl-
He/H2O
1-pyrroline N-oxide (DMPO)
§ H2O2 ~ constant across solutions
§ ●OH + ●OH → H2O2
(k = 5 x 109 M-1s-1)
§ DMPO + ●OH → DMPO–OH
(k = 4.3 x 109 M-1s-1)
§ H2O2 must be produced
exclusively in the gas phase
10
Myers, B. et al. Journal of Physics D: Applied Physics 54.14 (2021): 145202Isolating H2O2 origins using OH scavenger
He/O2 § H2O2 varies significantly between
solutions
§ Observed previously in phenol*
§ H abstraction by O from C-H
bonds
§ O enters liquid and reacts further
to form H2O2
§ OH or HO2 as precursor?
11
Myers, B. et al. J. Phys. D: Appl. Phys. 54.14 (2021): 145202 *Hefny, M. et al. J. Phys. D: Appl. Phys. 2016;49(40):404002.Comparison DMPO, TA, Phenol
§ Ring structures
§ H abstraction by O from C-H
bonds
§ O enters liquid and reacts further to
DMPO form H2O2
TA § When ring structures are present,
H2O2 production increases – ring
structures become part of the liquid
chemistry
Phenol 12From Ring Structures to Cysteine
§ Amino acid cysteine as simple model
§ He/H2O
§ up to 1 min.: DI ~ cys ~ cys + H2O2
§ 5 min: mock > DI >> cys x4.2
§ Cys consumed H2O2
§ Not if only H2O2 is present: short-
lived species necessary to initiate
reactions
§ He/O2
x2
§ DI water higher H2O2 concentration
§ Cys consumed H2O2
13
Stapelmann, K. et al. Journal of Physics D: Applied Physics 54 (2021): 434003Cysteine Modifications
§ Native cysteine
disappears
completely after 5
min He/H2O
§ Primary modification
He/H2O: cystine and
variations
§ Primary modification
He/O2: oxidation of
sulfur
14
Stapelmann, K. et al. Journal of Physics D: Applied Physics 54 (2021): 434003Cysteine Modifications – Origin of Species
§ Heavy water H218O
as liquid
§ He/H2O: more 18O
incorporated
§ 18O due to
hydrolysis, (V)UV or
metastable impact,
or 16OH H hopping
15
Stapelmann, K. et al. Journal of Physics D: Applied Physics 54 (2021): 434003Increasing complexity – buffer & medium
He/H2O He/O2
§ Cell culture medium (RPMI) and buffer (KPO4) used for plasma medicine
experiments influence H2O2 production for He/O2
§ Likely due to O interacting with organics in the medium 16Closer look
RPMI Medium
Glycine L-Isoleucine L-Tryptophan
L-Arginine L-Leucine L-Tyrosine
L-Asparagine L-Lysine L-Valine
L-Aspartic Acid L-Methionine
L-Cystine L-Phenylalanine Vitamins
L-Glutamine L-Proline Inorganic Salts
L-Histidine L-Serine Glucose
L-Hydroxyproline L-Threonine Glutathione
RPMI cell medium: a lot of components!
§ 3 amino acids with ring structures
§ 2 sulfur-containing amino acids 17What does it mean for plasma medicine?
Mock treatments to isolate effects – measure species in correct solution
He/O2 based mock treatment of Jurkat cells
x1.05
x12
H2O2 in RPMI
H2O2 in RPMI
Untreated
H2O2 in DI
Untreated
H2O2 in DI
18
Ranieri P et al. Applied Sciences. 2020;10(6):2025.What happens when cells are present?
§ Intercomparison study COST jet and nsp-DBD
§ Completely different setups, different chemistry, …
110
§ Cell viability as starting point
% Viability Normalized to Control
100
§ Jurkat cells (3!104 cells per well) 90
80
§ Incubated for 24 hr prior to assesing cell viability 70
60
§ Image cytometry 50
40
30
§ Conditions with comparable cell viabilities: 20
10
§ COST jet 2 min ~ nspDBD 45 Hz (10 s) 0
Gas Control 2 minutes 4 minutes 45 Hz 75 Hz
§ COST jet 4 min ~ nspDBD 75 Hz (10 s) COST-Jet nspDBD
19
Ranieri P et al. Applied Sciences. 2020;10(6):2025.nspDBD and COST jet
- Cell produced chemistry
Not only the cell viability is comparable, but also the mitochondrial
superoxide production in response to plasma treatment
COST jet 2 min ~ nspDBD 45 Hz (10 s)
16 110
% Viability Normalized to Control
100
14
90
12 80
MitoSOX (%)
10 70
60
8
50
6 40
4 30
20
2
10
0 0
Untreated 2 min 45 Hz Gas Control 2 minutes 4 minutes 45 Hz 75 Hz
COST-Jet nspDBD COST-Jet nspDBD 20
Ranieri P et al. Applied Sciences. 2020;10(6):2025.COST jet and nspDBD
- Liquid chemistry
225 12
Peroxide Concentration (µM)
11
200 Nitrite Concentration (µM) § As expected, the liquid chemistry is
Peroxide Concentration (µM)
10
Nitrite Concentration (µM)
175 9 completely different
150 8
7 § Long-lived species such as H2O2
125
6 and nitrite not (exclusively)
100 5
responsible for cell death
75 4
50 3
2
25 1
0 0
2 minutes 4 minutes 45 Hz 75 Hz
COST-Jet nspDBD
21
Ranieri P et al. Applied Sciences. 2020;10(6):2025.nspDBD - Liquid chemistry with cells
22
H. Mohamed et al. Cancers. 2021;13(10):2437.Conclusions
§ Organic matter becomes part of the chemistry
§ Different types of organic matter affect chemistry differently
§ By offering a target for reactive species / precursors for other long-
lived species (e.g. OH / H2O2)
§ By providing new precursors to form other species (e.g. H + HO2 /
H2O2)
§ Living cells actively contribute to liquid chemistry
§ By offering a target for reactive species
§ By uptake and neutralization of ROS
§ By releasing reactive species (TBD) 23Acknowledgements
Brayden Myers, María Herrera
Hager Mohamed, Eric Gebski, Quesada, Eleanor Lenker,
Fred Krebs, Vandana Miller Pietro Ranieri
DE-SC0021329
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