Transcript Document

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: [email protected]
Introduction
Study of interaction of small atmospheric discharge with living cells [1,2]
Use of Plasma Needle [3]
Results and discussion
Fluorescence measurements of radical concentration
9.0E+04
8.0E+04
7.0E+04
intensity (a.u.)
Effect on cell culture: cell detachment
6.0E+04
5.0E+04
4.0E+04
3.0E+04
2.0E+04
1.0E+04
Cell detachment in time. 1) control
sample, 2) 15 min. after treatment, 3) 1
hour after treatment, 4) 4 hours after
treatment.
position
0.0E+00
0
5
10
15
20
25
distance
NO (μM)
Calibration of fluorescence by NOR-1
From 10 µM curve flattens - saturation
9
2.5
8
2
6
radicals (μM)
Effect on tissue engineered skin: First test shows formation of vacuoles
and detachment
radicals (μM)
7
5
4
3
2
Cross section of tissue
engineered skin.
Control sample (left) and
sample treated for 5 min
(right).
Plasma applied  cells detach and remain alive
1.5
1
0.5
1
0
0
0
1
2
3
4
5
6
7
8
9
0
1
2
time (min)
3
4
5
6
distance (mm)
Linear time dependence
Distance: less than 1/r2 !
Radical concentration in µM range – fysiological concentration
Distance of tip needle-surface liquid > 2mm
radical concentration less than half
1.4
Material and methods
Cl
O
H
O
OCCH3
OH
radicals (μM)
1
1.5
1
0.8
0.6
0.5
0.2
0
0

Calibration with NO releaser NOR-1
Releases NO within 15 min after dissolving
Measurements performed with microplatereader
2
3
4
5
Position: less important
COH
O
ClH2C
ClH2C
Fluorescent probe before
and after activation and
reaction with radical
In situ picture
1
position (mm)
O
O
Cl
C OCCH3
O O
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
2
0.4
CM-H2DCFDA probe:
is dissolved in liquid
becomes fluorescent upon reaction with radical
Probe reacts to: H2O2, HO., HOO., ONOO- and NO
O
CH3CO
1.2
radicals (μM)
What causes this cell reaction? Is it caused by radicals?
Detect and measure radicals
2.5
6
0
0
0.2
0.4
0.6
0.8
added oxygen (%)
1
1.2
Extra oxygen, no radical increase
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.
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