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Fibrillar disruption by AC electric field induced

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DOI: 10.1016/j.bpc.2017.04.004

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 Biophysical Chemistry xxx (2017) xxx-xxx

Contents lists available at ScienceDirect

Biophysical Chemistry
journal homepage: www.elsevier.com

F
OO
Fibrillar disruption by AC electric field induced oscillation: A case study with human
serum albumin
Shubhatam Sena⁠ , Monojit Chakrabortyb⁠ , Snigdha Goleyb⁠ , Swagata Dasguptac⁠ ,⁠ ⁎⁠ , Sunando DasGuptab⁠ ,⁠ ⁎⁠

PR
a
Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur 721302, India
b
Department of Chemical Engineering, Indian Institute of Technology Kharagpur, Kharagpur 721302, India
c
Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India

ARTICLE INFO ABSTRACT

D
Keywords: The effect of oscillation induced by a frequency-dependent alternating current (AC) electric field to dissociate
Amyloid fibrils preformed amyloid fibrils has been investigated. An electrowetting-on-dielectric type setup has been used to ap-
Human serum albumin ply the AC field of varying frequencies on preformed fibrils of human serum albumin (HSA). The disintegration
AC electric field potency has been monitored by a combination of spectroscopic and microscopic techniques. The experimental
TE
Frequency
results suggest that the frequency of the applied AC field plays a crucial role in the disruption of preformed
HSA fibrils. The extent of stress generated inside the droplet due to the application of the AC field at different
frequencies has been monitored as a function of the input frequency of the applied AC voltage. This has been
accomplished by assessing the morphology deformation of the oscillating HSA fibril droplets. The shape defor-
mation of the oscillating droplets is characterized using image analysis by measuring the dynamic changes in the
shape dependent parameters such as contact angle and droplet footprint radius and the amplitude. It is suggested
EC

that the cumulative effects of the stress generated inside the HSA fibril droplets due to the shape deformation
induced hydrodynamic flows and the torque induced by the intrinsic electric dipoles of protein due to their con-
tinuous periodic realignment in presence of the AC electric field results in the destruction of the fibrillar species.

few experimental studies related to electric field induced conforma-

RR

1. Introduction
The response of proteins to different forms of exogenous perturba-
tions such as temperature [1–3], pressure [2], pH changes [4], chemi-
cal molecules/ions [5–9], laser excitation [10], and electromagnetic ra-
diation [11–14] is a topic of major research interest. The possible ef-
fects of external electric field stress on proteins has also been investi-
CO
tional switchover of proteins [26–28]. It may also be noted that most
studies are focused on electric field induced structural dynamics of the
native protein and the effect of electric field directly on the aggregated
protein fibrils have not been sufficiently explored. It is worth mention-
ing here that alternating current (AC) electric field has been utilized
in various biological applications e.g. fracture healing, tumor ablation,
and inhibition of cancerous cell growth [29,30]. Despite the involve-

gated [15–20], but most studies are theoretical and consider a molecu-
lar dynamic (MD) approach to examine the effects of static and pulsed
electric fields on the stability of different proteins. For example, MD
simulations have been used to investigate the helical to β-sheet confor-
mational transition of β-amyloid peptides on interaction with an elec-
tric field [15] whereas the generation of helical structure by disrupt-
ing native β-sheet conformation induced by an electric field has also
UN
ment in a variety of applications, the direct application of the AC elec-
tric field in the disintegration of amyloid fibrils has not yet been inves-
tigated. These facts reiterate the importance of studies focusing on the
influence of the external electric field on amyloid fibrils. The response
of amyloid fibrils under the stress induced by an AC electric field is thus
of interest.
Droplet-based microfluidics serves as a promising platform to ex-

been reported [17]. Some studies have also shown that the oscillating
external electric fields with certain frequencies have greater destabiliz-
ing effects on the secondary structure of the insulin chain-B than the
static electric field with the same strength [21–23]. A similar trend has
been observed in a MD simulation study of chignolin under an exter-
nal oscillating electric field [24]. Lugli et al. have conducted MD sim-
ulations to investigate the stability of Aβ fibrils under the perturbation
of an externally applied electric field [25]. However, there are very
perimentally analyze biochemical reactions using tiny droplets as con-
tainers of biological entities [31]. The basic principle of the technique
is based on the phenomenon commonly known as Electrowetting on
dielectric (EWOD), wherein electrical energy is used to alter the sur-
face energy and manipulate droplet shapes on a dielectric surface. The
technique has found considerable usage in a number of applications
starting from cell-based assays, protein analyses, and electronic cool-
ing etc. [32–35]. In this configuration, an insulating layer acting as a

⁎ Corresponding authors.
Email addresses: swagata@chem.iitkgp.ernet.in (S. Dasgupta); sunando@che.iitkgp.ernet.in (S. DasGupta)

http://dx.doi.org/10.1016/j.bpc.2017.04.004
Received 15 February 2017; Received in revised form 7 April 2017; Accepted 12 April 2017
Available online xxx
0301-4622/ © 2016 Published by Elsevier Ltd.
S. Sen et al.
dielectric is inserted between the liquid and the counter electrode to
prevent current flow. The principle of alteration of wetting properties of
the liquid by modifying the interfacial tension at the three phase contact
line induced by electrical stress is commonly known as electrowetting
[36]. However, as the electrical wetting tension weakly depends on the
polarity of the applied voltage, AC voltage may be used instead of di-
rect current (DC) voltage [37,38]. The AC electric field has additional
advantages including decrease in contact angle hysteresis [39,40], delay
in contact angle saturation [41], and decrease in biomolecular adsorp-
tion at the liquid-substrate interface [42]. The continuous occurrence
of wetting and de-wetting, caused by the application of AC voltage in
electrowetting, leads to oscillation of the droplet. The droplet oscilla-
tion induced by AC electrowetting in the low frequency regime leads to
shape deformation of the droplet, which in turn depends on the input
frequency of the AC electric potential. The induced oscillatory motion
can cause hydrodynamic flow inside the droplet leading to generation of
significant stress. The principle of AC electrowetting has found various
applications in biosensing, efficient mixing, micro-cooling, MALDI mass
spectrometry etc. [43–46]. However, to the best of our knowledge, no
study has been performed that takes advantage of AC electrowetting in
the disruption of preformed amyloid fibrils of protein and is the motiva-
tion of our present study.
The main goal of this study is to experimentally analyze the effect
of the application of an AC electric field on preformed fibrils of human
D
serum albumin (HSA), wherein the effect of field frequencies are probed
at a constant voltage. HSA, a physiologically important natively α-heli-
cal globular protein, has been shown to form fibrils with amyloid-like
characteristics under specific conditions [47,48] and thus serves as a
TE
suitable model protein to analyze the effect of the applied AC electric
field on the fibrils [49]. The effect of a static DC field with varying
strengths on preformed fibrils of HSA has previously been reported from
this laboratory. The study has shown that the HSA fibrils are signifi-
cantly disrupted on exposure to a static electric field having a strength
of ~ 8 × 106⁠ V m− ⁠ 1 for a period of 10 min [50]. In the present study,
EC
AC electric fields of various frequencies (5–100 Hz) at a fixed input volt-
age of the same electric field strength are applied on mature HSA fibril
droplets for different periods of time (1–6 min), keeping all other exper-
imental conditions unaltered. The effect of the application of the exter-
nal AC electric field of different frequencies on fibrils is monitored by
various biophysical techniques. Further to comprehend the stress gen-
RR
erated inside the oscillating HSA fibril droplet, responsible for the dis-
ruption of the amyloid fibrils, due to the application of the AC field, the
dynamic morphology change of the droplets at different frequencies is
characterized by monitoring the changes in different shape-dependent
parameters like droplet contact angle, droplet footprint radius, and the
amplitude. Further, to assess any thermal contribution to the observed
CO
structural changes, the temperatures of the sample droplets before and
after the application of electric field have also been measured.
UN
Fig. 1. Schematic diagram of the experimental set-up.
2
Biophysical Chemistry xxx (2017) xxx-xxx
2. Materials and methods
2.1. Materials
HSA and ThT were purchased from Sigma Chemical Co. (St Louis,
F
MO, USA) and used as received. ITO (Indium tin oxide, In2⁠ O3⁠ /SnO2⁠ )
coated glasses (surface resistivity of 30–60 Ω/sq) were purchased from
Sigma-Aldrich, St Louis, USA. Sylgard-184 (consisting polydimethyl-
OO
siloxane (PDMS) oligomer and cross linking reagent) was purchased
from Dow Corning, USA. All the other chemicals used were of analytical
grade and purchased from commercial sources and used without further
purification. All solutions were prepared using Milli-Q water (resistivity
~ 18.2 MΩ cm at 25 °C).
2.2. HSA fibril formation
PR
HSA was dissolved in Milli-Q water and the concentration mea-
sured spectrophotometrically using a molar extinction coefficient of
35,219 M− ⁠ 1
cm−
⁠ 1
at 280 nm [51]. HSA fibrils were prepared following
the reported standard procedure by incubating HSA (150 μM) at pH 7.0
(20 mM, Tris-HCl buffer) in the presence of 50% (v/v) ethanol at 37 °C
for 24 h [50].
2.3. Surface preparation
PDMS solution, to be used as a dielectric layer, was prepared by
mixing the elastomeric base and the cross linking agent in a weight ra-
tio of 10:1, followed by desiccation. A small amount of the PDMS was
then poured on the ITO coated glass substrate for coating of a thin layer
of PDMS over the glass using a spin coater (Süss MicroTec) which was
spun at 400 rpm for 30 s initially followed by at 4500 rpm for 70 s and
cured at 95 °C overnight. To increase surface durability and hydropho-
bicity, the substrate was further coated with a thin layer of Teflon.
The Teflon coating was performed by spin coating of Teflon solution
(5 wt%), dissolved in FC-40 solvent, at 3000 rpm for 30 s, followed by
curing at 130 °C for 20 min. The thickness of the PDMS and Teflon layer
were found to be 12.4 ± 0.4 μm and 23.4 ± 0.2 nm respectively, as mea-
sured by a profilometer. A portion of the substrate was left uncoated for
ground electrode connection.
2.4. Application of electric field
Using a controlled dispenser, a small droplet of the preformed HSA
fibrillar solution was placed over the prepared substrate and positioned
on a goniometer (Rame Hart, Germany) platform. The droplet and
substrate were electrically connected using the EWOD setup shown
in Fig. 1. One electrode was electrically grounded (connected to the
uncoated portion of the ITO glass) and AC
S. Sen et al.
electric potential was applied to the droplet by means of a platinum
wire electrode, dipped into the droplet. To apply the AC electric field
with tunable frequency, the electrodes were connected to a combined
AC signal generator (Agilent 33250A, 80 MHz waveform generator) and
an amplifier system (Tabor Electronics 9200A, High Voltage Wide Band
Amplifier). The AC power source was used to apply the alternating elec-
tric fields of various frequencies (5 to 100 Hz) on the HSA fibrillar so-
lution droplets for different exposure times (1–6 min). The peak to peak
value of the input AC voltage was maintained at 100 V. Thus the es-
timated electric field strength is found to be ~ 8 × 106⁠ V m−
⁠ 1
(the di-
electric layer thickness is ~ 12.5 μm). The temperature and the relative
humidity were maintained at 25 ± 0.5 °C and 35 ± 2% respectively dur-
ing the experiment.
2.5. Thioflavin T fluorescence study
ThT fluorescence of the electric field treated HSA solutions were
measured at room temperature using a Horiba Jobin Yvon Fluoromax-4
spectrofluorimeter. Aliquots, withdrawn from the sets of solutions, were
diluted using 20 mM Tris-HCl buffer of pH 7.0 to achieve final protein
and dye concentrations of 2 μM and 10 μM respectively. The excitation
wavelength was set at 450 nm, and the emission spectra collected from
470 nm to 600 nm. Slit widths for both excitation and emission were
D
set at 5 nm with an integration time of 0.3 s. All spectra were corrected
with respect to the corresponding blank spectra. Native HSA was found
not to affect ThT fluorescence under the experimental conditions. As a
control, a reference experiment on the application of the electric field
TE
on only ThT without fibrils was performed where no change in ThT flu-
orescence intensity was observed. All the experiments were performed
at least in triplicate.
2.6. Circular dichroism (CD) spectroscopy
EC
Far-UV CD spectra of HSA solutions were acquired using a Jasco-815
automatic recording spectrophotometer at room temperature. A quartz
cuvette of 0.1 cm path length was used. CD spectra were accumulated
in the wavelength range of 190 to 240 nm at a scan rate of 50 nm/min.
Protein concentrations for each measurement were kept at 2 μM. At
RR
least three scans were averaged to obtain one spectrum. The protein sec-
ondary structure content was determined using the online DICHROWEB
server [52].
2.7. Fourier transform infrared (FTIR) spectroscopy
CO
FTIR spectra of the HSA fibrillar samples were obtained using a
Thermo Nicolet 6700 FTIR spectrometer equipped with a zinc selenide
(ZnSe) attenuated total reflectance (ATR) accessory, a deuterated
triglycine sulfate (DTGS) detector and a KBr beam splitter. An aver-
age of 256 scans, for each spectrum, was recorded in the region of
4000–400 cm− ⁠ 1
with a 4 cm−⁠ 1
resolution. Control spectra were ac-
quired under the same experimental conditions and subtracted from
UN
the corresponding spectra. The amide I band region was subjected to
second derivative and Gaussian curve fitting to measure the total area
and area corresponding to each secondary structure component in the
1700–1600 cm− ⁠ 1 region. Secondary structure parameters were evalu-
ated using the method described earlier [53].
2.8. Turbidity assay
The absorbance at 350 nm of the HSA fibrillar solutions, diluted
with 20 mM Tris-HCl buffer of pH 7.0, were measured before and af-
ter the application of the AC electric field at varying frequency using
a Shimadzu-2450 UV–vis spectrophotometer. Each spectrum was cor-
rected with respect to the corresponding blank. All experiments were
performed at least in triplicate.
Biophysical Chemistry xxx (2017) xxx-xxx
2.9. Dynamic light scattering (DLS) assay
The changes in the aggregate sizes of HSA fibrillar solution were de-
termined before and after the application of alternating electric field
at varying frequency using a Malvern Nano ZS instrument employing
F
a 4 mW He-Ne laser (λ = 632.8 nm) and equipped with a thermostatic
sample chamber. All the solutions, diluted properly with buffer, were fil-
tered through 0.45 μm pore size micro-filters (Whatman International,
OO
Maidstone, UK). All measurements were performed at 298 K. The hy-
drodynamic diameter was obtained by a standard procedure using the
in-built software of the instrument.
2.10. Image analysis
The dynamic changes in the droplet shape during oscillation were
PR
captured using a high speed camera (model V641, Phantom) which was
connected to an image processing computer to analyze the captured im-
ages. Special light source (Leica CLS150 LED) was used to brighten the
captured images (setup shown in Fig. 1). The images, extracted during
oscillations at different frequencies, were captured using the Phantom
camera control (PCC, version 2.2) software at 2000 fps. The dynamic
changes in the droplet contact angle, radius of the droplet footprint and
the height of the droplet, from their equilibrium values were evaluated
from the extracted images using ImageJ (version 1.47), DROPimage Ad-
vanced (version 2.5.02) and ImagePro-Plus (version 6.0) software.
2.11. High resolution transmission electron microscopy (HRTEM)
The sample solutions were diluted and applied to carbon coated cop-
per grid. Negative staining was achieved via (1%, w/v) aqueous solution
of uranyl acetate. Samples were then air dried overnight and images
were acquired via a JEM 2100 high resolution transmission electron mi-
croscope operating at an accelerating voltage of 200 kV.
2.12. Fluorescence microscopy
For fluorescence imaging, 10 μL of each sample solution was incu-
bated with 5 μL of 1 mM ThT to achieve the required staining. Images
were captured using a Leica DM 2500 M microscope equipped with a
fluorescence attachment. Filter cube no. 2 (Leica I3 11, 513, 878, BZ:
01) was used for ThT excitation and emission. The images were ob-
tained with a Leica DFC 310 FX camera attached with the microscope.
All images were acquired at 10 ×/0.25 (N PLAN EPI).
3. Results and discussion
3.1. ThT binding assay
ThT, a fluorescent dye widely used for optical-based monitoring of
amyloid fibrillation, exhibits an enhancement in the fluorescence in-
tensity of the emission peak at 485 nm when excited at 450 nm, when
bound to amyloid fibrils [54]. After application of the AC electric field
for 5 min, samples were immediately supplemented with ThT and spec-
tra were recorded. The corresponding ThT fluorescence intensities at
485 nm of HSA fibrillar solutions before and after treatment with the AC
field (exposure time 5 min) at different frequencies are shown in Fig. 2.
The results show that the ThT intensities at 485 nm for all the treated
solutions are lower than the control with a minimum at 20 Hz. This in-
dicates that the application of an AC electric field of 20 Hz leads to the
most effective disintegration of the amyloid fibrils (~ 75% reduction in
ThT intensity). Since the ThT intensity is positively correlated to the
extent of fibrillation the reduction points towards the lesser amount of
fibrillar species present in the solution. The possible reason behind the
greater effectiveness of the electric field applied at 20 Hz in the disrup-
tion of the fibrils is discussed later.
S. Sen et al. Biophysical Chemistry xxx (2017) xxx-xxx

significant increase in intensity for the initial 2 min followed by a de-

creasing trend in ThT intensity beyond 2 min. This result indicates an
initial fibrillar growth followed by destruction of the amyloid fibrils at
a later stage on continuous application of the AC electric field. Yagi et
al. have reported a similar trend in case of laser induced destruction of
Aβ (1–40) fibrils, where an initial explosive growth of fibrils is observed

F
apparently caused by formation of active ends due to cleavage of fib-
rils [55]. It may be speculated from the experimental observations that
similar active regions of fibrillar nature are generated during the initial

OO
period of application of the alternating electric field. The self-assembly
process leads to the reconstruction of fibrillar species that is manifested
by an increase in ThT intensity. However, the preformed amyloid fibrils
are destroyed beyond 2 min with continuous application of alternating
field. It is also clear from Fig. 3 that the change in fluorescence inten-
sity is insignificant beyond an exposure time of 5 min. In this context,
it is important to note that droplet oscillation is also significantly re-
duced after 5 min due to the contact angle hysteresis phenomenon, i.e.

Fig. 2. Change in ThT fluorescence emission intensity at 485 nm of HSA solutions be-
fore (control) and after treatment of AC electric field (exposure time 5 min) of different
frequencies (± 1–3% error is associated with each experimental value which is estimated
from at least three individual measurements).
To assess the effect of exposure time of electric field, HSA fib-
rils were subjected to the AC field at 20 Hz for different time pe-
PR
a significant difference between the advancing and receding contact an-
gle of the moving contact line [56]. This implies that droplet oscillation
plays an important role in the defibrillation of preformed amyloid fib-
rils. For subsequent experiments, the HSA fibril droplets are exposed to
alternating electric fields of varying frequencies for a period of 5 min.
ThT fluorescence experiments have also been performed after 24 h and
48 h of application of electric field with no discernible changes in ThT

D
riods and the ThT binding study performed immediately after with- fluorescence intensities observed when compared to the results obtained
drawal of the electric field. Fig. 3 shows an interesting time-depen- for experiments performed immediately after application of the electric
dent change in fluorescence intensity with a field (Fig. S1). This observation rules out the possibility of recurrence of
the fibrillar species even after withdrawal of the field.
TE
3.2. Conformational transition of secondary structure: circular dichroism
(CD) study

To investigate the relation between the frequency of electric field os-

EC

cillation and conformational changes of HSA, the far-UV CD spectrum

of the protein samples are acquired before and after exposure to the AC
field for 5 min at varying frequencies. Fig. 4a shows the CD spectra of
HSA fibrillar solutions before and after the application of the AC electric
field at 20 Hz (other spectra are not shown).
The spectrum of native HSA shows two minima at 208 nm (corre-
sponding to π → π* transition of carbonyl groups in polypeptide chains)
RR

and 222 nm (corresponding to the n → π* transition of the carbonyl

Fig. 3. Change in ThT fluorescence intensity of HSA fibrillar solutions showing the effect
of exposure time of the AC electric field applied at 20 Hz (± 1–3% error is associated with
CO
group) indicating predominance of an α-helical conformation [57]. HSA
fibrillar solutions show significant reduction in the intensity (less nega-
tive) of those two bands indicating considerable decrease in the helical
structure, with a concomitant increase in the β-sheet content [58]. The
observed α-helix to β-sheet conformational transition is previously es-

each experimental value, estimated from at least three individual measurements). tablished to be associated with protein aggregation process [59]. How-
ever as seen in Fig. 4a, there is found to be an increase in mdeg val-
ues (more negative) at those two regions after exposure to the AC
electric field
UN

Fig. 4. (a) Far-UV CD spectra of native HSA and HSA fibrillar solutions before and after the application of the AC electric field at 20 Hz for 5 min. (b) Relative percentage reduction in
β-sheet content of HSA solutions after application of electric fields at varying frequency with respect to control HSA fibrillar solution (± 1–3% error is associated with each experimental
value, estimated from at least three individual measurements).
S. Sen et al.
at 20 Hz. This observation indicates that the electric field exposure on
the HSA fibrils disrupt the β-sheet structure along with restoration of the
native structure to a certain extent.
To estimate the secondary structure content of HSA, the online
DICHROWEB server has been used. Fig. 4b shows the relative % reduc-
tion in β-sheet content of the samples after application of alternating
electric fields at varying frequency with respect to the control HSA fib-
rillar solution. The data show a substantial reduction in the β-sheet con-
tent (~ 49%) at 20 Hz, which is consistent with the observations from
the ThT fluorescence study. It is thus apparent that the AC electric po-
tential affects the fibrillar structure of HSA, with the most prominent
disruption effect at 20 Hz. To support the results obtained from the CD
study, the secondary structural alterations of the protein induced by
the electric field at 20 Hz have also been monitored using FTIR spec-
troscopy and similar results are obtained (Fig. S2). The information re-
garding the secondary structural contents of HSA fibrillar solutions be-
fore and after application of AC electric field at 20 Hz, as obtained from
CD and FTIR study is summarized in Table 1.
3.3. Turbidity assay
Turbidity assays are frequently used in protein aggregation experi-
ments because intense light scattering, caused due to the self-associa-
D
tion of the elongated fibrils into the mature fibril networks, is reflected
in the increased turbidity of the samples i.e. higher absorbance value at
350 nm [58,60]. Fig. 5 represents absorbance values (350 nm) of HSA
fibrillar solutions before and after application of AC electric fields of
TE
varying frequency. The fibrillar solution, without the application of elec-
tric field, shows the maximum absorbance value, as expected. However,
consistent with the ThT fluorescence and the CD studies, significant
reduction (~ 78%) in absorbance is observed for 20 Hz. This marked
reduction in the turbidity value indicates the absence of aggregated
oligomers in the solution. The AC electric field applied at 20 Hz is able
EC
to disintegrate the HSA fibrillar structure most effectively, corroborat-
ing the results from the ThT binding and CD studies.
3.4. Aggregate size distribution: dynamic light scattering study
RR
In order to gain information regarding the size of the protein aggre-
gates present in the HSA fibrillar solutions before and after application
of alternating electric fields of different frequency, dynamic light scat-
tering measurements were performed. Fig. 6 shows the changes in the
mean hydrodynamic diameter of the aggregates present in the HSA fib-
rillar solutions. The size distribution histograms for HSA fibrils before
and after application of the AC electric field at 20 Hz are also presented
CO
in Fig. S3. It is apparent that the unfolding of HSA leading to amyloid
fibrillation is marked with the formation of considerably large aggre-
gates. However as evident from the trend, when the HSA fibrillar so-
lution is subjected to the electric field of different frequencies, distinct
change in the size of the aggregates, with the most significant reduction
at 20 Hz suggesting the presence of smaller species of HSA, is observed.
Thus the DLS result, in agreement with previous observations, indicates
UN
that application of the alternating field at 20 Hz causes maximum dis-
ruption of the HSA fibrillar aggregates.
Table 1
Comparison of secondary structural contents of HSA fibrillar solutions before and after application of alternating electric field at 20 Hz, as obtained from CD and FTIR study.
CD study
HSA fibril HSA fibril@20 Hz
α-Helix 35.7 ± 1.3% 44.5 ± 2.7%
β-Sheet 44 ± 2.1% 22.4 ± 1.8%
β-Turn 12 ± 0.3% 17 ± 1.2%
Random 8.3 ± 0.2% 16.1 ± 1.7%
Biophysical Chemistry xxx (2017) xxx-xxx
3.5. Frequency dependent oscillation of fibril droplet
In electrowetting, internal flows are generated inside a sessile
droplet when an AC electric potential is applied. However, the charac-
teristics and origin of the flows are found to be dependent on the ap-
F
plied frequency [61]. While the high-frequency (around and more than
35 to 256 kHz) electro-thermal flow is caused by the penetration of elec-
tric field into the liquid droplet, the low-frequency (around 10 Hz to
OO
15 kHz) hydrodynamic flow is due to the movement of capillary waves
from the contact line towards the apex of the droplet [62]. In the pre-
sent study, the effect of AC electric field has been investigated in the
low frequency range of 5–100 Hz, indicating a low-frequency flow in-
side the HSA fibrillar droplets and hence the direction of the flow in-
side the droplet may be expected to be of similar pattern, as observed
earlier for low frequency AC electrowetting [62]. It has also been es-
tablished that the low-frequency hydrodynamic flow is generated due to
PR
the oscillation of the droplet interface and the oscillation results from
the time-varying oscillatory component of the AC electrical force con-
centrated on the edge of the droplet [31,62]. A representative slow mo-
tion video of a HSA fibril droplet oscillating at a frequency of 20 Hz
is given in the Supplementary information (Video S1). Additionally, a
series of images extracted at characteristic times from the video of the
HSA fibril droplet oscillating at a frequency of 20 Hz is presented in Fig.
7.
The oscillation of the droplet surface induced by the low frequency
AC electrowetting results in the shape deformation of the droplet, which
in turn is a function of the input frequency of the AC field [45]. To assess
the optimum effectiveness of the frequency of 20 Hz towards the de-
struction of HSA fibrils, it is necessary to analyze the shape deformation
of the HSA fibril droplets during oscillations at different input frequen-
cies of the applied AC electric field. The change in the shape of the oscil-
lating droplet is quantitatively characterized by image analysis from the
extracted images of real-time oscillation videos (at 2000 fps) recorded
at different frequencies. The analysis is performed by measuring the dy-
namic changes in the shape dependent parameters such as contact an-
gle, contact radius (the radius of the droplet footprint), and amplitude
(difference in the height of the droplet in absence and presence of the
electric field) from their equilibrium values at each frequency and are
shown in Fig. 8. Additionally, the data for the maximum changes in the
parameters for a control experiment with the droplets without fibrils
(i.e. only the buffer) are presented in Fig. S4. The control experiment
indicates that the maximum deformation of the droplet takes place in
the frequency range of 10–20 Hz with slightly higher changes at 10 Hz.
However, the input frequency for maximum shape deformation of the
oscillating droplet containing the fibrils is obtained at 20 Hz. Therefore
it may be inferred that the presence of HSA amyloid fibrils marginally
affects the resonance frequency, while maintaining the basic physics of
the process.
3.6. Morphological and distribution aspect: high resolution transmission
electron microscopy and fluorescence microscopy
Further to corroborate the results obtained from the biophysical
techniques and also the image analysis, two microscopic techniques
namely high resolution transmission electron microscopy (HRTEM) and
fluorescence microscopy are used to monitor the morphology and dis-
tribution of HSA fibrils before and after the application of AC elec-
tric field of varying frequency. HRTEM images
FTIR study
HSA fibril HSA fibril@20 Hz
33.5 ± 1.6% 41.2 ± 2.3%
56 ± 2.8% 31 ± 1.6%
7.5 ± 0.4% 15.8 ± 0.7%
3 ± 0.1% 12 ± 0.6%
S. Sen et al. Biophysical Chemistry xxx (2017) xxx-xxx

of the HSA fibrillar solutions, before and after application of the alter-
nating electric field at different frequencies, are obtained. The corre-
sponding image of HSA fibrils (Fig. 9a) before application of the elec-
tric field shows an intense fibrillar network with complex branching,
whereas a substantial breakdown of the fibrillar structure into small ag-
gregates (non-fibrillar species) is observed for the solution exposed to

F
the AC electric field at 20 Hz (Fig. 9b). HRTEM images of HSA fibrillar
solutions at other frequencies are given in Fig. S5.
On a similar note, the ThT-stained fluorescence microscopic image

Fig. 5. Absorbance values at 350 nm of HSA fibrillar solutions before (control) and after
application of AC electric field of varying frequency for 5 min (± 1–3% error is associated
OO
of the HSA fibrillar solution in the absence of an electric field shows an
intense fluorescence suggesting an abundance of HSA fibrils (Fig. 10a).
However, after the treatment of the AC field at 20 Hz, the solution ex-
hibits a noticeable reduction in fluorescence intensity with almost no
residual fibrillar species suggesting a significant disruption of fibrillar
aggregates (Fig. 10b). This implies that the exposure of the fibrils to an
AC electric field at a frequency of 20 Hz causes significant disintegra-
tion of mature HSA fibrils.

PR
with each experimental value, estimated from at least three individual measurements).

3.7. Disruption of mature HSA fibrils by AC field

In an earlier report from this laboratory, it was shown that the max-
imum disintegration of fibrils takes place on application of a DC elec-
tric field strength of ~ 8 × 106⁠ V m− ⁠ 1 for 10 min [50]. In the present

study, the preformed HSA fibrils are exposed to AC electric field of vary-

D
ing frequency at the same field strength of ~ 8 × 106⁠ V m− ⁠ 1 (exposure

time only 5 min) keeping all the other experimental conditions unal-
tered. Results indicate an optimal frequency of 20 Hz where significant
destruction of fibrils is observed, as established from the experimental
TE
observations with respect to HSA fibrillar solution that acts as the con-
trol (~ 75% attenuation in ThT intensity, ~ 49% and ~ 45% reduction
in β-sheet content as obtained from CD and FTIR studies respectively).
The external AC field of that specific frequency (20 Hz) is found to in-
flict more damage to the HSA fibrils compared to the DC electric field
of the same strength. In addition it is also found to be associated with
EC

a faster destabilization of the fibrillar structure as lower exposure times

Fig. 6. Changes in the mean hydrodynamic diameter of the aggregates present in the HSA
fibrillar solutions before (control) and after application of the AC electric field of varying
frequency for 5 min (± 1–3% error is associated with each experimental value, estimated
from at least three individual measurements).
RR
are required compared to the previous study (5 min vs 10 min). The-
oretical studies show a similar trend where oscillating electric field is
found to have a more destabilizing effect on protein than a static field
of same effective strength [21–24]. The rapid change in the direction of
electric field is assumed to be more damaging to the secondary structure

of protein than the fixed electric field.

The higher effectiveness of the AC field can also be attributed to
the stress generated inside the oscillating droplet due to induced in-
ternal flow in AC electrowetting. The electrowetting phenomenon in-
duced by the electrical
CO
UN

Fig. 7. A series of images of a HSA fibril droplet oscillating at a frequency of 20 Hz at characteristic times.
S. Sen et al.
D
TE
Fig. 8. Variation in the maximum change in the shape dependent parameters of the HSA fibril droplet as a function of frequency: (a) contact radius, (b) contact angle and (c) amplitude
(± 2–4% error is associated with each experimental value, estimated from at least three individual measurements).
EC
RR
CO
Fig. 9. HRTEM images of HSA fibrillar solutions obtained (a) before and (b) after the application of AC electric field at 20 Hz for 5 min (scale bars represent 200 nm).
stress, commonly known as Maxwell stress, is concentrated at the three
phase contact line [63]. In AC electrowetting, oscillation of a droplet
is believed to be related to the time-harmonic component of Maxwell
stress, while the other time-independent mean component is responsi-
ble for the mean change in the apparent contact angle [31,63]. Thus
UN
the AC electric field induces an internal circulatory motion inside the
droplet due to the periodic wetting and dewetting, which leads to the
shape dependent oscillation of the droplet [62,63]. Consequently a sig-
nificant dynamic morphology change induced by the application of
AC field is believed to cause a considerable stress leading to destruc-
tion of the fibrils. In earlier reports also, there are evidences of the
role of an externally applied hydrodynamic stress significantly affect-
ing and eventually damaging the wall structure of cells leading to ei-
ther cell deformation or death [64,65]. From Fig. 8, the optimal fre-
quency for maximum change in the shape dependent parameters of
the droplet is obtained at 20 Hz i.e. when the frequency is of 20 Hz,
the droplet oscillation is most effective leading to maximum shape
deformation. Thus, the AC electric field at 20 Hz induces the maxi
7
Biophysical Chemistry xxx (2017) xxx-xxx
F
OO
PR
mum dynamic morphology change which in turn results in the gener-
ation of significant stress inside the HSA fibril droplets and is respon-
sible for the maximum disruption of the fibrils. As discussed earlier,
the induced internal flow inside the droplet is generated by the oscil-
lation of the droplet surface in low frequency AC electrowetting, not
by the electric field distribution inside the droplet because the elec-
tric field does not penetrate the droplet in the low-frequency range
(up to 15 kHz) [62]. On the basis of a earlier report [41], a schematic
of the distribution of the electric field lines around a droplet is pre-
sented in the Supplementary information (Fig. S8). The electrical stress
is hence prevalent only at the surface of the HSA fibril droplets. It is
also established earlier that the surface-active amyloid fibrillar species
have a propensity to accumulate at the air-water interface [66,67] ren-
dering them highly concentrated at the interfaces and thus are di-
rectly affected by the AC electric field that causes significant deforma-
tion in the droplet interface and as a result get disaggregated. The ab-
sence of an electric field distribution inside the droplet excludes the
possibility of Joule heating which has been observed only for very
S. Sen et al.
Fig. 10. ThT-stained fluorescence microscopic images of HSA fibrillar solutions acquired (a) before and (b) after the application of the AC electric field at 20 Hz for 5 min (scale
bar = 200 μm).
high frequency flows that are dominated by the electric field distribu-
tion inside the droplet [31,63]. Thus it is unlikely that there are any
temperature effects to the observed structural changes in this case. This
has been verified by measuring the temperature of the HSA droplets be-
fore and immediately after exposure to the electric field at 20 Hz for
D
5 min. The temperature distributions of the droplets have been cap-
tured by an infra-red (IR) thermal imaging camera FLIR SC5000 with
a temperature sensitivity of 0.020 °C through a software (ALTAIR: Ver-
sion 5.80.016) attached to the IR camera (experimental setup illustrated
TE
in Fig. 1). The corresponding temperature distributions of the droplets
(represented by the circular region) are shown in Fig. 11.
The observation indicates an average temperature increase of only
0.7 °C (21.6 °C to 22.3 °C) on application of the electric field which is
not significant enough to cause any structural variations in the sam-
ple. According to the theory of electrowetting, the shape deformation
EC
induced droplet oscillation does not depend on the dielectric prop-
erty of the solution in the range of frequency and voltage studied
herein [62]. It may be added that as there is no heating ef
RR
CO
UN
Fig. 11. Temperature distribution of the HSA fibril droplets (represented by the circular
region) acquired (a) before and (b) after exposure to the AC field at 20 Hz.
Biophysical Chemistry xxx (2017) xxx-xxx
F
OO
PR
fect due the AC electrowetting induced oscillation only natural evapora-
tion would take place which alters the contact radius and height of the
droplet marginally, resulting in a volume change of less than 1% in the
time scale of the experiment (5 min). However, as a control experiment,
a droplet of the fibrillar solution is allowed to evaporate naturally, in
absence of the electric field. An aliquot is extracted and subjected to ThT
fluorescence analysis. The absence of any change in the fluorescence in-
tensity relative to the initial fibrillar solution rules out the possibility
of any effect of evaporation during the process. It may also be noted
that the droplet volume will not affect the results in the present case,
as the Bond number (ρgL3⁠ /σ), which signifies the relative magnitude of
the gravitational force to that of the surface tension force, of the system
is found to be well below unity [Bo = 9.86 × 10− ⁠ 4 where ρ is the bulk
density of the system (950 kg/m for the solution used herein); L is the
3
⁠
characteristic length of the system (1.52 mm, the radius of the droplet
with 15 μL volume used in the experiments) and σ is the surface tension
of the solution (30 mN/m for the solution used)] [35]. A value less than
unity signifies the predominance of surface forces over gravity ensuring
that the droplet volume is not a factor influencing the results. Thus the
shape dependent AC voltage induced oscillation, responsible for the dis-
ruption of the fibrils, is solely due to the electro-modulated alterations
of the interfacial tension at the solid liquid interface.
Additionally, understanding the effect of AC field on preformed fib-
rils needs a consideration of attributes for the structural stability of the
amyloid fibrils. It has been established earlier that apart from hydropho-
bic and aromatic interactions, electrostatic interactions also play a cen-
tral role in amyloid fibril formation at the higher pH value form the
isoelectric point by stabilizing the structure in the hydrogen-bonding or
fibre axis direction and drive the anti-parallel arrangement of the ex-
tended β-strands within a β-sheet [68]. To delineate the role of elec-
trostatic effect, HSA aggregates, formed by incubating native HSA at
pH 5, are subjected to the AC field of 20 Hz and monitored using dif-
ferent techniques (details in the Supplementary information). The re-
sults indicate only indiscernible changes in the aggregates upon appli-
cation of the AC field at 20 Hz. A representative video for the exper-
iment with the droplet of amorphous aggregates, included in the Sup-
plementary section (Video S2), shows only slight shape oscillation. It is
observed that the changes in the shape dependent parameters for the
droplet containing HSA amorphous aggregates are insignificant as com-
pared to that for the fibrillar droplet, when subjected to the AC field
at 20 Hz. The comparison has been illustrated in Table S1. The notice-
able reduction in the oscillation of the droplet, containing the amor-
phous aggregates, may be due to the deposition of the cluster of large
amorphous aggregates at the solid-liquid interface of the droplet. It is
to be mentioned that transition of native HSA molecules to amorphous
aggregates at pH 5.0 is also found to involve significant increase in vis-
cosity and lowering of solubility, as reported earlier [48] . These result
in an additional resistance to the contact line movement [69], which
can be clearly seen from the captured video. Thus, the experimental ob-
servations indicate that the shape oscillation of the droplet containing
HSA aggregates is not significant enough to produce sufficient hydro
S. Sen et al.
dynamic flows inside the droplet to disrupt the aggregates. It is also
worth mentioning that HSA has almost no net charge at pH 5, which is
close to the pI (4.9) of the protein [70] . Thus the absence of the elec-
trostatic effect (at pH 5) and the reduced hydrodynamic flow may be
ascribed as reasons to the unsuccessful application of electric field to
disrupt the aggregates. On the other hand, the zeta potential of HSA
fibrils at pH 7.0 is found to be − 34 ± 0.76 mV, indicating HSA is sub-
stantially negatively charged. This observation points towards the role
of the electrostatic effect in the disintegration of HSA amyloid fibrils.
A similar observation has been reported earlier where an external elec-
tric field is shown to decrease the β-sheet in higher oligomers of β-amy-
loid peptide having a net negative charge [25]. Along with the charged
side chains and oppositely charged terminal residues, dipoles of peptide
backbone of the protein are responsible for the electrostatic effect [27].
Thus, the protein possesses a dipole moment which is likely to be af-
fected by electric field. This dipole moment allows the protein to inter-
act strongly with the applied electric field, which causes the protein to
realign its secondary structure dipoles along the field direction leading
to alteration in protein structure. Previous theoretical studies have given
evidence that a reduction in β-sheet content of protein is possible under
the action of electric field due to the rearrangement of the anti-paral-
lel dipoles of the β-sheet in the presence of the external electric field
[17,25]. It can be speculated that the intrinsic electric dipoles of protein
go through continuous periodic realignment with respect to the direc-
D
tion of alternating electric field causing a continuous stretching and re-
laxation of the protein molecules [22]. Thus the torque induced by the
electric field may cause significant perturbation of various noncovalent
interactions responsible for the stability of fibrillar structure, leading to
TE
the disruption of fibrils. The probable involvement of the hydrodynamic
flows and the electrostatic forces [71] in the disintegration of the HSA
fibrils is shown in Fig. 12.
However, to further reveal the importance of the hydrodynamic
flow in disruption of amyloid fibrils, an experiment has been conducted
where significantly larger volume (200 μL) of HSA fibrillar solution is
EC
exposed to the AC electric field at 20 Hz in an eppendorf tube, under
the same experimental condition in a slightly modified setup. The setup
is shown in Fig. S9, where an eppendorf is cut into two parts and the
upper part is attached to the surface of the Teflon coated ITO glass. The
post-experiment analysis of the treated fibrillar solution with ThT fluo-
rescence spectroscopy and CD spectroscopy indicate only ~ 15% reduc-
RR
tion in ThT intensity and only ~ 8% decrease in β-sheet content respec-
tively (Fig. S10). It may be mentioned here that ~ 75% reduction in ThT
intensity and ~ 49% decrease in β-sheet content were observed for the
same experiment in a droplet assay. This indicates the significance of
the stress generated due to the shape induced hydrodynamic flow inside
the droplet for fibrillar disruption. In the eppendorf, where the hydrody-
CO
namic flow is absent, only electrostatic effect is operative causing lesser
(compared to the droplet assay) disintegration of the fibril. Thus it may
be assumed that the overall destruction of the HSA fibrillar species in
the droplet assay is primarily driven by the stress generated inside the
HSA fibril droplets due to the shape deformation induced hydrodynamic
flows and partially by the electrostatic effect.
UN
Fig. 12. A schematic diagram depicting the probable involvement of the hydrodynamic
flows and the electrostatic forces responsible for disruption of the HSA amyloid fibrils.
Biophysical Chemistry xxx (2017) xxx-xxx
4. Conclusions
It has been shown that oscillation induced by application of an al-
ternating electric field (at a fixed strength of ~ 8 × 106⁠ V m−⁠ 1) signif-
icantly aids in the disintegration of preformed HSA fibrils. It is conclu-
F
sively established that the frequency of the electric field plays a crucial
role in the process. The disintegration process has been monitored by a
combination of various spectroscopic and microscopic techniques. The
OO
experimental results reveal that the external AC field of a specific fre-
quency (20 Hz) has the most destabilizing effect on preformed mature
HSA fibrils (~ 75% decrease in ThT intensity, ~ 49% and ~ 45% re-
duction in β-sheet content as obtained from CD and FTIR study respec-
tively, with respect to control HSA fibrillar solution). The field expo-
sure time-dependent ThT fluorescence study indicates an initial fibril-
lar growth followed by the destruction of the amyloid fibrils. The maxi-
mum dynamic morphology change due to the shape deformation of the
PR
HSA fibril droplet is also found at a frequency of 20 Hz, characterized
by the maximum changes in shape-dependent parameters (contact an-
gle, droplet footprint radius, and amplitude), corroborating the exper-
imental observations from the biophysical techniques. The study has
demonstrated that at the lower frequency range of the applied AC field,
the temperature change is insignificant and thereby does not influence
the results. It is suggested that the shape deformation induced hydrody-
namic flows inside the HSA fibril droplets in the presence of the AC field
mainly augments the destruction of the fibrillar species. The application
of the AC field causes the rapid and continuous alignment of the sec-
ondary structure dipoles of the protein along the electric field direction
and consequently the induced torque results in significant perturbation
of the non-covalent interactions, responsible for the stability of fibrils.
Thus, the present study provides new physical insights into the disrup-
tion of the amyloid fibrils through exploitation of frequency-dependent
oscillation induced by the alternating electric field.
Acknowledgements
The authors gratefully acknowledge the financial support provided
by the Indian Institute of Technology Kharagpur, India (Grant num-
ber: IIT/SRIC/ATDC/CEM/2013-14/118, dated 19/12/2013). The au-
thors are also thankful to Central Research Facility, IIT Kharagpur for
the instrumental facilities.
Note: The authors declare no competing financial interest.
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