Supplemental Material
Combined effect of protein and oxygen on reactive oxygen
and nitrogen species in the plasma treatment of tissue
Nishtha Gaur, Endre J. Szili, Jun-Seok Oh, Sung-Ha Hong, Andrew
Michelmore, David B. Graves, Akimitsu Hatta, and Robert D. Short
Contents
1:
Experimental methods: Phosphate buffered saline; Preparation of gelatin target;
Preparation of poly(vinyl alcohol) target; Preparation of RONS reporters;
Helium cold atmospheric plasma jet treatment; Microplate reader
measurements; UV absorption spectroscopy; Atomic force microscopy;
Statistical analysis
Page 2
2:
Calibration curves to quantify H2O2 and NO2-
Page 6
3:
Topography and roughness of the gelatin surface after CAP exposure
Page 7
4:
Topography and roughness of the PVA surface after CAP exposure
Page 8
5:
Helium-assisted transport of 5,6-carboxyfluorescein (CF) through gelatin and
PVA
Page 9
6:
Time dependency of H2O2 and NO2- generation in PBS directly exposed to CAP
Page 10
7:
Measurement of molecular, ground state O2 in PBS by UV absorption
spectroscopy
Page 11
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1: Experimental methods
Phosphate buffered saline.
Phosphate buffered saline (PBS) was prepared by dissolving one PBS tablet (Sigma-Aldrich, catalogue
number P4417) in 200 ml deionized water (18.2 MΩ.cm) purified through a Millipore Direct-Q system
(catalogue number ZRQ50PO30). PBS was filtered through a 0.2 µm syringe filter.
Preparation of gelatin target.
A 5% (w/v) gelatin film was prepared by dissolving 50 mg ml-1 gelatin powder (Sigma-Aldrich, catalogue
number G1890) in PBS. A hot water bath at 40°C with continuous stirring for 15-20 min was used to
dissolve the gelatin powder. Gelatin films of 1 mm thickness were prepared by pouring 9 ml into an 85 mm
diameter (plastic) petri dish lined with parafilm® (Beemis, catalogue number PM-996). The plate was gently
agitated to enable the solution to evenly spread over the bottom of the dish, whilst taking precaution not to
induce air bubbles into the solution. The gelatin was allowed to set at 4˚C for 12 h before use. Square
sections of approximately 10 x 10 mm were cut using a scalpel and removed with a polytetrafluoroethylene
tweezer, taking care not to tear the gelatin. The gelatin film was self-supporting.
Preparation of poly(vinyl alcohol) target.
A 10% (w/v) poly(vinyl alcohol) (PVA) target was prepared by dissolving 100 mg ml-1 of PVA powder
(Sigma-Aldrich, catalogue number 363065) in PBS. A hot water bath at 200°C with continuous stirring for
45-50 min was used to dissolve the PVA powder. The solution was allowed to cool to 90°C for 30 min.
PVA films of 1 mm thickness were then prepared as described (above) for gelatin.
The gelatin / PVA sections were placed over the top a 96-well plate (Costar®, catalogue number 3596) so
that the films completely covered the top of each well and that it remained in contact with the PBS
containing the reactive oxygen and nitrogen species (RONS) reporters, inside the wells.
Preparation of RONS reporters.
The RONS reporters were prepared as follows:
Hydrogen peroxide (H2O2) detection – A mixture of 18.5 mM ortho-phenylenediamine (OPD) powder
(Sigma-Aldrich, catalogue number P9029) and 4 µg ml-1 horseradish peroxidase (HRP) powder (SigmaAldrich, catalogue number P6782) was dissolved in PBS.
2
Nitrite (NO2-) detection – Griess reagent powder (Sigma-Aldrich, catalogue number G4410) was dissolved
in PBS at a concentration of 50 mg ml-1.
Hydroxyl radical (OH•) detection – Terephthalic acid powder (Sigma-Aldrich, catalogue number 185361)
was dissolved in PBS at a concentration of 500 mM.
Broad spectrum reactive oxygen species (ROS) detection – 2,7-dichlorodihydrofluorescein diacetate
(DCFH-DA) powder (Sigma-Aldrich, catalogue number D6883) was prepared by dissolving the powder in
ethanol to a concentration of 2 mM; the solution was stored at -20°C until use. Ester hydrolysis was induced
by adding 500 µl of stock DCFH-DA to 2 ml of 10 mM NaOH and incubating for 30 min at 25°C in the
dark. The DCFH (deacetylated) solution was then neutralised by adding 10 ml of PBS. The solution was
stored in the dark until use and discarded if not used within one day.
The RONS reporters were used at a volume of 400 µl per well of a 96-well plate. This volume ensured the
solution always remained in contact with the gelatin or PVA targets that were placed over the wells.
Helium cold atmospheric plasma jet treatment.
A helium cold atmospheric plasma (CAP) jet was used in this study. It comprised a standard glass pipette
tube with an inner diameter of 1 mm, which was surrounded by two external hollow electrodes separated at
a distance of 10 mm. The end of the tube protruded 1 mm out from the bottom electrode. The CAP was
operated with 100 ml min-1 of helium at an applied voltage potential of 5.5 kVpeak-peak and a frequency of 10
kHz. These operational conditions produced a plasma plume of 5 mm in length into the ambient atmosphere.
The plasma jet was operated in the ambient air and the 96-well plate containing the RONS reporter
solutions, with / without the targets, was positioned using a laboratory scissor jack. The platform of the
scissor jack was plastic so the 96-well plate or gelatin / PVA targets were not grounded. The flow rate of the
helium gas was controlled with mass flow controllers and power was supplied with a custom-built unit as
described elsewhere (Al-Bataineh et al, Plasma Process. Polym., 8: 2011, 695). Treatment was carried out at
a distance 1 mm between the end of the glass capillary tube (of the CAP assembly) and the top of the RONS
reporter solution or the gelatin / PVA targets. To analyse treatment from the neutral helium gas (i.e. plasma
off), the treatment was carried out using the same flow rate of helium but with no applied voltage. It was
found that neutral helium treatment of 10 min did not change the colour or fluorescence of any of the
reporters following direct treatment of the RONS reporter solutions or through the gelatin / PVA targets.
Therefore, the control used for data normalization of reporter was the “as-prepared” RONS reporter solution.
3
Microplate reader measurements.
A 100 µl volume of the test solutions was transferred to a separate 96-well plate to measure the absorbance
or fluorescence of the test solutions. For Griess reagent, absorbance measurements were recorded at 492 nm
and 450 nm for OPD / HRP. Fluorescence of oxidized DCFH was recorded at excitation of 485 nm and
emission of 520 nm with a gain of 650. Where mentioned in the paper, the data was normalized by dividing
the test RONS reporter solutions by the as-prepared RONS reporter solution.
UV absorption spectroscopy.
UV absorption spectroscopy (Hitachi, U-3900) was used for measuring the O2 level of PBS. PBS has an
extremely low transmittance in the UV range below 210 nm. For achieving a reasonable UV transmittance
level, 100 m optical path cuvettes (cells with detachable windows, Hellma, 106-QS) were used. A 100 l
aliquot of the test PBS was diluted with 1 ml deionized water just before the UV measurement. To confirm
UV absorption spectra of O2 molecules in PBS, high purity O2 gas was purged into 3 ml of PBS in a quartz
cuvette (Hellma, 101-QS). The transmittance of PBS was measured before and after the O2 treatment. The
same method was used for 400 l of PBS in 96-well plate uncovered and covered gelatin.
Atomic force microscopy.
Atomic force microscopy (AFM) was used to determine the elastic modulus, surface roughness and
qualitatively analyse the surface topography of the gelatin and PVA films. The films were set onto rigid
silicon wafers to make it compatible for AFM analysis. An NT-MDT NTEGRA SPM with a 100m piezo
scanner in non-contact mode was used for imaging of the surface. The scanner was calibrated using 1.5 m
grids with a height of 22 nm. The tips used were silicon nitride NSG03 gold coated tips with a tip radius of
10 nm. Image analysis, including roughness calculation, was performed using NT-MDT Nova software.
AFM was also used to obtain force–distance curves of the gelatin in air to determine their elastic modulus.
The spring constant of the cantilevers was measured using the Sader method (Sader et al, Rev. Sci. Instrum.,
70: 1999, 3967) and was in the range of 1 – 3 N m-1. Force–displacement curves were obtained by retracting
the tip 1000 nm from the surface and then approaching the surface at 100 nm s-1 until 200 nm beyond the
contact point. 5 force–displacement curves were collected at different points for each sample. Assuming that
the modulus of the silicon nitride tip (~150 GPa) is much greater than that of the gelatin, for the case of a
parabolic tip pressing on a flat surface, the Hertz model can be used to derive the following expressions
(Choukourov et al, Plasma Process. Polym., 9: 2012, 48):
 = F2/3
4
where  is the deflection of the surface, F is the applied force and the fitting parameter  is related to
Young's modulus, E, by:
3(1 − 𝜇 2 )
𝐸 = 3/2 1/2
4𝛼 𝑅
where  is Poisson's ratio (assumed to be 0.3) and R is the radius of the tip.
Statistical analysis.
Statistical analysis was performed on selected datasets using an unpaired Student t-test assuming unequal
variances. A p value of less than 0.05 was considered to be significantly different (95% confidence).
5
2: Calibration curves to quantify H2O2 and NO2Calibration curves were constructed using known concentrations of H2O2 and sodium nitrite (NaNO2,
Sigma-Aldrich, catalogue number S2252). A 10 µl volume of known concentrations of H2O2 and NaNO2
were mixed with 90 µl of PBS containing the OPD / HRP or Griess reporters for H2O2 and NaNO2,
respectively (from SI-1). After incubation for 30 min at 25°C in the dark, the absorbance of the test solution
was measured on the miroplate reader as described in SI-1.The calibration curves for H2O2 and NaNO2 are
shown in Fig. S1. The calibration curves were established around the same time of the real experimental
measurements (within days) to avoid any drift in experimental variation over time.
FIG S1. Calibration curves of (a) H2O2 and (b) NaNO2 that were used to quantify H2O2 and NO2- in PBS.
6
3: Topography and roughness of the gelatin surface after CAP exposure
FIG S2. Surface topography of typical (a) as-prepared gelatin target; after exposure to neutral helium flow
(no plasma) for (b) 1, (c) 5 and (d) 10 min; and after exposure to the helium CAP jet for (e) 1, (f) 5 and (g)
10 min. The number in the top left corner of each image represents the typical roughness (root mean square)
value of each respective surface.
7
4: Topography and roughness of the PVA surface after CAP exposure
FIG S3. Surface topography of a typical PVA target. The number in the top left corner of the image
represents the typical roughness (root mean square) value of the PVA surface.
8
5: Helium-assisted transport of 5,6-carboxyfluorescein (CF) through gelatin and PVA
FIG S4. Helium-assisted delivery of CF through gelatin and PVA. The targets were placed over the top of an
empty 96-well plate. A 100 l drop of 50 mM CF was dispensed on the gelatin or PVA surface. The centre
of the CF spot was treated with the neutral helium flow at 1000 ml min-1 at a distance of 1 mm between the
end of the glass tube and top of the CF spot. After treatment, the targets were turned over so that the
underside faced up. A 400 l volume of PBS was spotted onto the target to mix the CF dye that had
penetrated through the target. The FI intensity of CF was measured on the microplate reader at excitation of
485 nm and emission of 520 nm. The FI was normalized against the FI measured in the as-prepared PBS.
9
6: Time dependency of H2O2 and NO2- generation in PBS directly exposed to CAP
Direct CAP exposure resulted in significant evaporation of the solution in the 96-well plate for exposure
times longer than 1 min. Therefore, shorter exposure times of 5 – 60 s were used to illustrate the time
dependency of H2O2, NO2- and OH• generation in PBS. The result is shown in below in Fig. S5.
FIG S5. CAP generation of H2O2, NO2- and OH• directly in uncovered PBS (without gelatin or PVA).
Measurement of (a) H2O2, (b) NO2- and (c) OH•. Measurements were taken after 30 min incubation at 25°C
in the dark. The concentration of (a) H2O2 and (b) NO2- in (b) was determined from calibration curves in SI2. In (c) the data are presented as normalized fluorescence intensity (FI) because the absolute OH•
concentration could not be quantified from a calibration curve. The dashed line in (c) indicates a threshold,
below which all numbers are effectively zero.
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7: Measurement of molecular, ground state O2 in PBS by UV absorption spectroscopy
FIG S6. Detection of O2 in uncovered PBS and in PBS covered with gelatin. The (a) UV absorption
spectrum of PBS was taken after purging with molecular, ground state O2. The absorption maximum for O2
in PBS was determined to be 190 nm. The peak absorbance at 190 nm is shown in (b) for PBS (uncovered or
covered with the gelatin target) after exposure to the He CAP jet. The data show that a higher concentration
of O2 is detected in PBS covered with the gelatin target, cf. uncovered PBS.
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