Effects of helium atmospheric pressure plasma jet on biological
molecules
I. Topala, A.V. Nastuta, V. Pohoata, N. Dumitrascu
Plasma Physics Laboratory, Faculty of Physics, Alexandru Ioan Cuza University, Iasi, Romania
Abstract: Atmospheric pressure plasma jet is a useful tool nowadays in plasma
medicine. This plasma source is used for clinical applications such as
cauterization, coagulation or wound healing. In most of the clinical cases when
the plasma jet acts on tissues, we are dealing with a very complicated reaction
mechanism. Cells and proteins are surrounded by a liquid environment, and
plasma can modify the properties of all these three components with different
kinetics. In order to know more about the mechanisms at the interface plasma –
biological tissues, we employed the helium plasma jet to modify biological
molecules (test protein: albumin) in solid (thin film) state and in aqueous
solution. Also effects of plasma action on biological liquids were monitored (e.g.
pH modification, local temperature modification).
Keywords: plasma medicine, helium plasma jet, proteins
1. Introduction
2. Materials and methods
Atmospheric pressure plasma technologies have a
growing importance in many technologies indented
to be applied in medicine, biology and health.
Subjects that involve plasma physics and technology
(e.g. living tissue treatment or wound healing, cancer
cell apoptosis, blood coagulation, sterilization and
decontamination) are nowadays in study in many
laboratories. A continuously increasing number of
scientific publications or reports on these subjects
are published in the last years [1-3].
The atmospheric pressure plasma jet (APPJ) used in
this study is based on a cylindrical barrier discharge
with external electrodes. We have used a quartz
tube, 4 mm inner diameter and 6 mm outer diameter,
to generate an atmospheric pressure plasma jet
(Fig. 1).
Using the principles of corona discharge, dielectric
barrier discharge and microdischarges many
atmospheric pressure plasma sources are being
developed, under well know names such as plasma
needle, plasma pencil, plasma gun or atmospheric
pressure plasma jet (APPJ). Parameters of
atmospheric pressure plasma sources (e.g. specific
temperatures and concentration / distribution of
charged species, gas temperature, concentration and
distribution of reactive species, life time of active
species generated in the plasma volume and at
interfaces) are spread over large domains of values.
Figure 1. Long exposure time photography of the helium
plasma jet focused on a human finger and a sketch that contains
the principal elements used to generate the plasma jet.
Aluminum tape electrodes (10 mm width for the
power electrode and 4 mm width for the ground
electrode) are wrapped on the external surface of the
tube, being separated by a 10 mm gap. Helium
(spectral purity) is flowing inside the quartz tube
with electronically controlled flow rate.
High voltage monopolar square pulses are applied
on the powered electrode with variable repetition
rate, amplitude and pulse width using an
amplification chain: signals supplied by a function
generator are amplified with x1000 factor by a HV
power source (PD07016, Trek). The results
presented here are obtained with positive HV pulses
of 2 kHz frequency. The discharge current and
applied voltage are monitored using voltage dividers
and current probes, connected to a digital scope
(Fig. 2). The plasma jet has typical lengths of few
centimeters for He flow rates up to 5 L/min. The jet
can be focused on living tissues or any type of
substrates. Using an optical fiber and a
photomultiplier (R955) we monitored the light
emission in the vicinity of the substrate (Fig. 2).
Function of working frequency the precise time of
plasma action onto various substrates can be
measured and controlled in this way.
transferred into the wells of a microtiter plate. Each
protein containing well was exposed 1 min to the
helium plasma jet, 1.5 cm distance from tube’s edge,
and then the protein was transferred into distilled
water in order to obtain a solution of 1 mg/mL
concentration. Second derivative analysis of UV and
fluorescence spectra was used in order to detect
changes in the amino acids environment or protein
structural modifications.
In situ study of the plasma jet effect on albumin
films was done inside the sample chamber of the
UV-Vis spectrophotometer. The albumin film was
obtained on a quartz window by spotting droplets of
protein solution and then evaporating the solvent.
The protein films was then introduced into the
measurement beam and exposed continuously to the
action of the plasma jet, oriented at 45° with respect
to quartz window. Absorption spectra in the UV
range were taken each 30 s.
3. Spectroscopic studies of plasma
modified proteins
The UV absorption spectra of the thin albumin film
present significant absorbance values in the 190 –
250 nm range. The action of helium plasma jet leads
to a decrease of the absorbance for the entire studied
wavelength range (Fig. 3). This is related to the mass
loss and thickness decrease by protein removal
under the plasma action [4-6]. Previous investigation
of our plasma jet revealed the presence of highly
reactive chemical species based on oxygen (e.g. O,
OH) at the interfaces with biological samples.
Figure 2. Typical applied voltage and discharge current
waveform; total light emission from a finger surface exposed to
the action helium plasma jet (1.5 cm from tube’s edge).
To study the effects of helium atmospheric pressure
plasma jet on biological molecules, we employed a
model protein, the Bovine Serum Albumin - BSA
(Sigma Aldrich). For UV absorption (Thermo
Evolution 300 spectrophometer) and fluorescence
studies (Perkin-Elmer Lambda 3 spectrofluorimeter)
the protein powder was weighted and than
Figure 3. Time dependent UV absorption spectra of the albumin
film under continuous action of the plasma jet.
The absorbance decrease was found to be influenced
by the applied voltage (Fig. 4). For a given value of
the applied voltage the absorbance decreases
linearly.
Blue-shifts of the peaks in the second derivative
absorption spectra are associated usually the with the
aromatic residue exposure to a more polar
environment, due to protein unfolding. As shown in
Fig. 5, the peaks corresponding the tryptophan,
tyrosine and phenylalanine have nearly no shift after
protein exposure to helium plasma jet. This can be
associated with no major protein unfolding.
However the second derivative of fluorescence
emission spectra shows some differences between
native and plasma modified proteins (Fig. 6). It’s
worth to stress that these changes are otherwise
unobservable in the original emission spectra [7].
Figure 4. Time dependence of the absorbance at 195 nm for
increasing values of the applied voltage.
The removal speed is higher as the applied voltage
increases. This can be explained by the increase of
dissipated power in the discharge volume and the
efficient production of oxygen based reactive
species.
UV absorption spectroscopy is a common
experimental technique used to provide information
on major structural changes in proteins. Aromatic
residues, such as tryptophan (Trp), tyrosine (Tyr),
and phenylalanine (Phe) present significant
absorbance in the UV spectral range (200–300 nm).
The molecular environment and the amino acids
integrity give rise to changes in intensity of the
plasma modified proteins absorption spectra (Fig. 5).
Figure 5. Second derivative of the UV absorption spectra, for
native and plasma modified albumin in solution.
Figure 6. Second derivative of fluorescence spectra, for native
and plasma modified albumin in solution, λexc = 285 nm.
The tryptophan from BSA in distilled water shows
for the native protein bands at 337, 359 and 375 nm.
The spectrum of the plasma modified protein present
a red-shift for the first band, now located at 339 nm,
and blue-shifts for the last two bands, now located at
352 and 371 nm. This suggests the presence of
partially unfolded states of proteins, due to subtle
tertiary structure changes.
These preliminary investigations on the structure of
plasma modified proteins suggest that apart from
protein destruction, plasma action on protein
powders can induce also subtle structural
modifications. These modifications can occur during
plasma treatment or after the protein transfer in
solution, due to reorganization phenomena. Further
investigations are necessary to understand these
effects, the kinetics of modification and the structure
of plasma modified proteins.
5. Conclusions
References
Helium atmospheric pressure plasma jet represents a
good candidate to be used as plasma source for
medical applications. Experiments regarding the
plasma effects on supramolecular biological systems
like proteins offer a better understanding of
mechanisms that are behind the benefic effects in
plasma medicine. Beside protein removal, the main
effect for long exposure duration, changes in the
protein structure can be induced by the short
exposure time. This can affect the protein function.
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The
financial
support
from
the
Grant
POSDRU/89/1.5/S/63663 is highly acknowledged.
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