2015_Influence of surface charge on the rate, extent, and structure of adsorbed

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Journal of Colloid and Interface Science 460 (2015) 321–328
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Journal of Colloid and Interface Science
journal homepage: www.elsevier .com/locate / jc is
Influence of surface charge on the rate, extent, and structure of adsorbed
Bovine Serum Albumin to gold electrodes
http://dx.doi.org/10.1016/j.jcis.2015.08.055
0021-9797/� 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding author at: Department of Chemical Engineering, Carnegie
Mellon University, Pittsburgh, PA 15213, USA.
E-mail addresses: bbeykal@andrew.cmu.edu (B. Beykal), herzberg@bgu.ac.il
(M. Herzberg), yoramo@bgu.ac.il (Y. Oren), mauter@cmu.edu (M.S. Mauter).
Burcu Beykal a, Moshe Herzberg b, Yoram Oren b, Meagan S. Mauter a,c,⇑
aDepartment of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA
b Zuckerberg Institute for Water Research, Ben-Gurion University of the Negev, Sede Boqer 84990, Israel
cDepartment of Engineering & Public Policy, Carnegie Mellon University, Pittsburgh, PA 15213, USA
g r a p h i c a l a b s t r a c t
a r t i c l e i n f o
Article history:
Received 30 May 2015
Revised 15 August 2015
Accepted 22 August 2015
Available online 24 August 2015
Keywords:
Protein adsorption
Bovine Serum Albumin
Electrochemical QCM-D
Cyclic voltammetry
a b s t r a c t
The objective of this work is to investigate the rate, extent, and structure of amphoteric proteins with
charged solid surfaces over a range of applied potentials and surface charges. We use Electrochemical
Quartz Crystal Microbalance with Dissipation Monitoring (E-QCM-D) to investigate the adsorption of
amphoteric Bovine Serum Albumin (BSA) to a gold electrode while systematically varying the surface
charge on the adsorbate and adsorbent by manipulating pH and applied potential, respectively. We also
perform cyclic voltammetry-E-QCM-D on an adsorbed layer of BSA to elucidate conformational changes
in response to varied applied potentials. We confirm previous results demonstrating that increasing mag-
nitude of applied potential on the gold electrode is positively correlated with increasing mass adsorption
when the protein and the surface are oppositely charged. On the other hand, we find that the rate of BSA
adsorption is not governed by simple electrostatics, but instead depends on solution pH, an observation
not well documented in the literature. Cyclic voltammetry with simultaneous E-QCM-D measurements
suggest that BSA protein undergoes a conformational change as the surface potential varies.
� 2015 Elsevier Inc. All rights reserved.
1. Introduction
Understanding andmanipulating interactions between colloidal
particles and solid substrates is of critical importance to engi-
neered systems for coatings, biomedicine, food processing, and
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322 B. Beykal et al. / Journal of Colloid and Interface Science 460 (2015) 321–328
environmental science. Extensive prior research has investigated
the role of colloidal properties [1–6], solution characteristics [7–
11], surface materials and patterning [12–15], as well as external
fields [16–20] in controlling the rate, extent, and structure of the
adsorbed layer.
While protein adsorption to non-polarized surfaces has been
thoroughly examined experimentally and theoretically [21–28],
considerably less effort has been dedicated to understanding the
influence of electric fields on the electrostatic interactions of pro-
teins, or other amphoteric macromolecules, with a charged sub-
strate. In the presence of an electric field, the rate of attachment,
the total mass deposition, and the conformation of colloids and
macromolecules may be altered due to enhanced electrostatic
interactions with the electrode surface [29–34].
To probe these effects, Ying et al. [20] investigated the adsorp-
tion of Human Serum Albumin (HSA) on gold electrodes for four
different applied potentials via electrochemical and ellipsometric
methods. They report enhanced protein adsorption on both posi-
tive and negatively charged surfaces. Moulton et al. [19] used radi-
olabeling to determine the total mass adsorbed on the gold
electrode for HSA and immunoglobulin G (IgG) proteins at 3 differ-
ent applied potentials, while also investigating the electrochemical
behavior of the adsorbed layer via cyclic voltammetry. In contrast
with the results reported by Ying et al., Moulton et al. reported that
negatively charged electrodes hindered the adsorption of nega-
tively charged HSA. Brusatori et al. [35] employ optical waveguide
lightmode spectroscopy (OWLS) to study Bovine Serum Albumin
(BSA) and horse heart cytochrome C protein adsorption kinetics
to charged surfaces, finding that both positively and negatively
charged proteins demonstrated increased adsorption with positive
applied potential, though only the rate of BSA adsorption
increased. And, finally, Benavidez and Garcia [36] demonstrated
that under applied voltages in excess of 0.5 V, high protein concen-
trations, certain pH values, and select ionic strengths, the polariz-
ability of pre-adsorbed BSA leads to enhanced electrostatic
interactions with dissolved proteins and ultimately to multilayer
adsorption. Their experimental work is limited, however, in
addressing the effect of applied potential during the initial stages
of protein-electrode interaction.
These contrasting and sometimes counterintuitive results sug-
gest the need for additional investigation into the factors affecting
protein adsorption in the presence of an electric field, particularly
during the initial stages of interaction. In particular, optical and
radiolabeling methods would benefit from complimentary mass-
based adsorption measurements via the Electrochemical Quartz-
Crystal Microbalance with Dissipation Monitoring (E-QCM-D)
method. E-QCM-D offers the additional benefit of capturing the
viscoelasticity of the adsorbed layer on an electrode, which is of
critical importance in understanding the structure of the adsorbed
layer and the conformational changes of the protein during cyclic
voltammetry. Xie et al. [37] studied the QCM–EIS system for
simultaneous measurement of electrochemical and acoustic impe-
dance and showed that this novel method can generate accurate
and interference-free results. Thus, we adopt a similar system
where we use the E-QCM-D to quantify adsorption to charged
surfaces.
In this work, we seek to isolate the contributions of electrostatic
forces originating at both the protein and the electrode and to
investigate their relative importance in electroadsorption and pro-
tein conformation. We investigate the rate and extent of adsorp-
tion of amphoteric BSA, a model soft protein, to a gold electrode
by systematically varying the surface charge on the adsorbate
and adsorbent by manipulating pH and applied potential, respec-
tively. We also perform cyclic voltammetry-E-QCM-D on an
adsorbed layer of BSA to elucidate conformational changes in
response to varied applied potentials.
2. Materials and methods
2.1. Measuring the zeta potential to determine the charge of Bovine
Serum Albumin as a function of pH
The charge on Bovine Serum Albumin (BSA) molecules are
tracked by measuring the zeta potential of 12 samples of 10 g/L
BSA (Amresco) suspended in 10 mM NaCl solution at different pH
values using Malvern ZetaSizer Nanoseries ZSP. The pH of each
sample is adjusted by titrating either with 0.1 M HCl (Fisher Scien-
tific, Certified ACS Plus, 36.5–38.0% w/w) or 0.1 M NaOH (Fisher
Scientific, Certified ACS).
2.2. Measuring adsorption to charged surfaces
We measure the adsorption of BSA to a gold surface using a
Quartz-Crystal Microbalance with Dissipation Monitoring (QCM-D;
Q-Sense E4,Biolin Scientific). QCM-D is a nanogram sensitive
instrument for assessing macromolecule–surface interactions. A
piezoelectric quartz crystal is oscillated at its resonance frequency
as mass is adsorbed to the surface of the crystal. Resulting changes
in the frequency and dissipation of the crystal provide real-time
insight into adsorbed mass and the viscoelastic and inertial
characteristics of the deposited layer in real-time [38–41].
The frequency and the dissipation data can be converted to
units of adsorbed mass per unit area by using the Sauerbrey or
viscoelastic models. Sauerbrey equation relates the change in
frequency to change in mass, as in Eq. (1),
Dm ¼ �C
n
Df ð1Þ
where n is the overtone number (n = 1, 3, 5, 7, . . .) and C is the mass
sensitivity constant which has the value of �17.7 Hz ng/cm2 for a
5 MHz crystal [25,38]. This relation holds under three major
assumptions [38]. First, the adsorbed mass follows the motion of
the sensor, thus the adsorbed film is rigid. Second, the adsorbed
mass is small relative to the mass of the sensor. And third, the
adsorbed mass is evenly distributed on the crystal.
If the adsorbed film layer has viscous and elastic contributions
to the frequency change, the Sauerbrey relation no longer holds.
Instead, the Voigt based viscoelastic film model can be incorpo-
rated in the calculation of the adsorbed mass. Voigt based model
assumes a homogenous viscoelastic layer with uniform thickness
covering the surface of the piezoelectric quartz crystal [41–44].
As described by Höök et al. [42], the adsorbed film is in contact
between the gold coated electrode and a semi-infinite Newtonian
liquid. Using this model, Voinova et al. [44] derived the relation-
ship between the viscoelastic properties of the adsorbed mass
and the QCM-D response as in Eqs. (2) and (3),
Df � � 1
2pqoho
g3
d3
þ h1q1x� 2h1
g3
d3
� �2 g1x2
l21 þx2g21
( )
ð2Þ
DD � 1
pfqoho
g3
d3
þ 2h1 g3d3
� �2 g1x
l21 þx2g21
( )
ð3Þ
where qo and ho is the density and the thickness of the crystal,
respectively, g3 is the viscosity of the bulk liquid, q3 is the density
of liquid, d3 is the viscous penetration depth of the shear wave on
bulk liquid, and x is the angular frequency of oscillation. q1, g1,
l1, and d1 represents the density, viscosity, shear elasticity and
the thickness of the adsorbed layer, respectively. In the present
work, we use the Q-tools (Q-sense) software to model the total
mass adsorption.
QCM-D allows users to monitor the changes in frequency and
dissipation as a function of surface charge when coupled with a
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Fig. 1. Zeta potential of 1 wt% BSA dissolved in 10 mM NaCl over the pH range of 2
and 11.
B. Beykal et al. / Journal of Colloid and Interface Science 460 (2015) 321–328 323
potentiostat. This configuration is titled an electrochemical QCM-D
(E-QCM-D) where the gold-coated quartz crystal is the working
electrode, a platinum surface serves as the counter electrode, and
Ag/AgCl electrode is the reference electrode [45]. These are con-
nected to the potentiostat (BioLogic, SP-50) to maintain a constant
voltage between the working and the reference electrodes
throughout each experiment. The electrochemistry module has
an internal volume of approximately 100 lL.
Before starting the experiment, 14 mm AT-cut piezoelectric
quartz crystals (Q-sense, QSX 338, Gold with Ti adhesion layer)
with a fundamental resonant frequency of 4.95 MHz are treated
with UV/ozone for 10 min. Later, the sensors are placed in an
ammonium hydroxide (Alfa Aesar, ACS, 28.0–30.0% NH3) and
hydrogen peroxide (BDH Chemicals, 30%) solution (1:1:5 H2O v/v/v)
at 348 K for 5 min. Each sensor is then washed with 18.2 MO
deionized water and dried using N2 gas. Finally, the sensors are
treated again with UV/ozone for 10 min. In each experiment, we
begin by flowing deionized water over the gold crystal for 600 s
to equilibrate the system. Next, we run 10 mM NaCl through the
E-QCM-D for 1200 s. After this step, we run BSA dissolved in
10 mM NaCl solution for 3600 s. In the last 1800 s, we rinse
the system with 10 mM NaCl, followed by rinsing in DI water.
Volumetric flow rate is held constant throughout the experiment
at 150 lL/min.
2.3. Preparation of gold-coated quartz crystals for cyclic voltammetry
(CV) experiments
We prepare the gold electrodes used in the CV experiments by
first cleaning the gold-coated quartz crystals (Q-sense, QSX 338,
Gold with Ti adhesion layer) as described in Section 2.2. For the
CV testing of the uncoated crystal, the gold-coated quartz sensor
is equilibrated with 10 mM NaCl solution for 1800 s. For the CV
testing of the BSA coated crystal, we flow deionized water over
the pristine gold crystal for 600 s. Next, we run 10 mM NaCl
through the E-QCM-D for 1200 s. After this step, we run BSA dis-
solved in 10 mM NaCl solution for 3600 s. In the last 1800 s, we
rinse the system with 10 mM NaCl to remove the excess protein
in the flow chamber.
3. Results and discussion
This work characterizes the influence of electrostatic interac-
tions on the adsorption of a model amphoteric biomacromolecule,
Bovine Serum Albumin (BSA), to externally polarized surfaces. To
do so, we systematically vary surface charge on both the adsorbate
and adsorbent by manipulating pH and applied potential, respec-
tively. Finally, we perform a simultaneous cyclic voltammetry-E-
QCM-D experiment and sweep the voltage on the BSA-coated gold
electrode to investigate the possible conformational changes in
BSA with varying surface potential.
3.1. Amphoteric behavior of Bovine Serum Albumin
Amino acids, the building blocks of proteins are amphiprotic,
meaning that they can both accept and donate a proton via reac-
tion with their amine (–NH2) and carboxylic acid (–COOH) func-
tional groups, respectively. We leverage this amphoteric behavior
to manipulate charge on the BSA macromolecule, while preserving
its secondary structure [46].
The zeta potential of Bovine Serum Albumin (BSA) changes sig-
nificantly over the pH range, 11–2, as shown in Fig. 1. The isoelec-
tric point is approximately 4.4, which is consistent with the range
(4.5–5.0) reported in literature [47–50]. At pH values lower
than 4.4, BSA carries a net positive charge, while at pH values
above 4.4, the zeta potential is negative and BSA carries a net
negative charge.
Although secondary structure is preserved across the range of
pH values investigated in this study, there are some changes to
the tertiary structure of the protein. For the following experiments
at pH = 6.529, BSA is in the normal form (N-form) with
[26,46,51,52] the most compact conformation. For experiments
conducted at pH = 2.846, BSA is in an extended form (E-form), in
which the BSA protein acquires maximum dimension and asym-
metry [26,46,51,52].
3.2. Bovine Serum Albumin adsorption in the absence of an electric
field
A wide range of methods have been used to study protein
adsorption to solid surfaces. Optical methods, including ellipsome-
try [20,27,53–55], optical waveguide lightmode spectroscopy
(OWLS) [27,56–60], and surface plasmon resonance (SPR) [61–
63] provide high quality data on the average thickness of the
adsorbed layer. Unfortunately, these techniques are limited by
the properties of the surface (i.e. only highly transparent surfaces
can be used in OWLS [27]), provide little insight into the mechan-
ical properties of the adsorbed mass, and cannot measure the
water or any other solvent that might be coupled to the protein.
Alternatively, acoustic techniques, such as QCM-D, allow for a
wider range of surfaces and allow for monitoring of the viscoelas-
ticity of the adsorbed mass, but cannot distinguish between the
adsorbed protein and the coupled water [28,42].
In this study, we are particularly interested in studying the vis-
coelasticity of the adsorbed mass, which provides insights into the
conformational changes that occurunder varying electrode polar-
ity and applied potentials. As a result, we rely on mass based meth-
ods for measurement of the adsorbed layer.
We performed BSA adsorption experiments in the absence of an
electric field to assess the importance of the solution concentration
on the total mass adsorbed and on the viscoelasticity of the
adsorbed ‘‘wet mass”, or the mass of the protein plus the mass of
the coupled water. Our results for BSA adsorption to an uncharged
gold surface are consistent with previous work [64] reporting a
positive correlation between BSA solution concentration and
adsorbed mass. For solution concentrations of 0.02 g/L, 0.20 g/L,
and 1.00 g/L, the change in dissipation is less than 1E-6, indicating
that the mass is rigidly adsorbed to the surface. We modeled each
of these three data sets using the Sauerbrey relation (Eq. (1)), and
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Fig. 2. Frequency, dissipation and total mass adsorption of BSA at open circuit conditions. (A) Frequency data for 1.00 g/L, 0.20 g/L and 0.02 g/L from QCM-D measurement
with no applied electric potential. (B) Dissipation data for 1.00 g/L, 0.20 g/L and 0.02 g/L from QCM-D measurements with no applied electric potential. (C) Amount of
adsorbed BSA per unit area with 3 different concentrations adsorbed to uncharged surface. The 7th overtone is reported for both the frequency and dissipation for all solution
concentrations.
Fig. 3. Adsorption of BSA as a function of electrode polarity and protein charge. (A) QCM-D data showing the change in frequency as a function of the adsorption of negatively
charged BSA to both positively charged (black) and negatively charged (red) gold surfaces. (B) Modeled adsorbed mass (BSA + coupled water) for BSA adsorption to positively
and negatively charged surfaces when BSA is suspended in a nearly neutral solution and is negatively charged. (C) QCM-D data showing the change in frequency as a function
of the adsorption of positively charged BSA to both positively charged and negatively charged gold surfaces. (D) Modeled adsorbed mass (BSA + coupled water) for BSA
adsorption to positively and negatively charged surfaces when BSA is suspended in an acidic solution and is positively charged. BSA concentration = 0.20 g/L in 10 mM NaCl
for all measurements. 7th overtone is reported. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
324 B. Beykal et al. / Journal of Colloid and Interface Science 460 (2015) 321–328
the estimates of adsorbed wet mass in ng/cm2 are plotted in
Fig. 2C.
After first rinsing with 10 mM NaCl solution, we perform a sec-
ond rinsing step with deionized water. This allows us to quantify
the mass of adsorbed ions, and we find that the mass decrease is
similar across the three experimental conditions. Additionally,
the change in adsorbed mass upon hydrated ion desorption closely
matches the change in adsorbedmass associated with hydrated ion
adsorption during the equilibration stage of each experiment at
t = 900 s. This is further supported by the cyclic voltammetry
results presented in Section 3.5. Fig. 5C.
3.3. Effect of protein charge and electrode potential on BSA adsorption
3.3.1. Negatively charged BSA
At pH = 6.529, BSA is negatively charged and in its N-form. As
expected from electrostatics, we observe a greater drop in fre-
quency when there is a positive applied potential than when there
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B. Beykal et al. / Journal of Colloid and Interface Science 460 (2015) 321–328 325
is a negative applied potential (Fig. 3A). This data is later converted
to total adsorbed wet mass using Eq. (1), shown in Fig. 3B, which
includes the adsorbed mass from both the protein molecule and
the coupled water.
There is also a significant difference in the rate of wet mass
adsorption to the positively and negatively charged surfaces. Fol-
lowing on Brusatori et al. [35], for the positive case, there is a dis-
tinct transport limited regime followed by an initial, linear surface
limited adsorption regime with an apparent adsorption rate con-
stant to the bare surface of 8.2E-5 cm s�1 (Fig. 4A). This initial lin-
ear region is then followed by an asymptotic region of the surface
limited regime. For the negative applied potential, however, there
is no evidence of distinct transport regimes, suggesting that the
electrostatic repulsion between the protein and the surface is dom-
inating the kinetics of adsorption.
3.3.2. Positively charged BSA
At pH = 2.846, BSA is positively charged and in an extended con-
formation. When the sensor is negatively charged, we expect to see
more adsorption to the gold surface due to electrostatic attraction.
This is confirmed in Fig. 3C, where the frequency shift is greater on
the negatively charged sensor than the positively charged surface.
Again, this frequency shift is converted to adsorbed wet mass in
Fig. 3D.
Interestingly, neither the total mass adsorbed nor the rate of
adsorption is as strongly influenced by the charge of the electrodes
when BSA is positively charged. Positively charged BSA
exhibits nearly instantaneous adsorption to both the negatively
and positively charged electrode surfaces (Fig. 3A), and there is
much less difference between the apparent adsorption rate
Fig. 4. Adsorption rate versus adsorbed mass curves for proteins (p) and surfaces (s) of v
linear region of the surface limited regime, the intercept of which provides the apparent a
back to the intercept. (A) Negatively charged BSA adsorption to a positively charged surfa
no distinctly different regimes noted for this case, and the blue hashed line is provided on
surface. (D) Positively charged BSA adsorption to a positively charged surface. BSA conc
(For interpretation of the references to color in this figure legend, the reader is referred
constants of 7.5E-5 cm s�1 (Fig. 4C) and 5.7E-5 cm s�1 (Fig. 4D),
respectively.
These observations may stem from the extended conformation
that BSA adopts under acidic conditions. Reduced total adsorption
may be a function of the BSA protein occupying a larger area on the
surface, a difference in the orientation of the ellipsoidal protein, or
the absence of protein clustering or multilayer adsorption [35,65].
Additional experimental work may be useful in helping to precisely
differentiate between wet mass adsorption and protein adsorption
under these conditions.
3.4. Effect of varying the magnitude of applied potential on BSA
adsorption
To differentiate between electrostatic attraction and other
attractive forces, we modulate the applied potential while main-
taining constant charge on the adsorbate. Electrochemical QCM-D
experiments for negatively charged BSA (pH = 6.787) at 0.20 g/L
under 7 different applied potentials are presented in
Fig. 5A and B. At larger magnitudes of positive applied surface
potential we observe the greatest mass adsorption, whereas at
the highest magnitude of negative charge we observe the lowest
adsorption. As expected, the total adsorbed mass approaches equi-
librium values that are proportional to both the magnitude and
sign of the applied potential, with the highest adsorption at
+0.5 V and lowest adsorption at �0.5 V.
This influence of magnitude is highlighted in the positive
applied voltage case. At +0.5 V, the electrostatic attraction between
the positively charged surface and the negatively charged BSA
molecule dominates over the EDL repulsion between the BSA
arying charge, corresponding to data presented in Fig. 3. Red solid lines refer to the
dsorption rate constant. Blue hashed lines are the extrapolation of this linear region
ce. (B) Negatively charged BSA adsorption to a negatively charged surface. There are
ly for visual reference. (C) Positivelycharged BSA adsorption to a negatively charged
entration = 0.20 g/L in 10 mM NaCl for all measurements. 7th overtone is analyzed.
to the web version of this article.)
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Fig. 5. Frequency, total mass adsorption (BSA + coupled water) and rate of mass adsorption under different constant applied voltage. (A) Frequency data (7th overtone) of
0.20 g/L BSA for varying electrode potential. (B) The effect of applied potential on total mass adsorption of 0.20 g/L BSA at pH of 6.787. (C) Rate of adsorption of BSA at different
electrode potentials.
326 B. Beykal et al. / Journal of Colloid and Interface Science 460 (2015) 321–328
molecules. However, as the magnitude of the surface charge is
decreased to +0.1 V, the EDL repulsion between the BSA
molecules starts to dominate over the electrostatic attraction
to surface, resulting in reduced total mass adsorption to gold
surface.
Unlike total adsorbed mass, however, the initial rate of BSA
adsorption does not appear to be proportional to the magnitude
of the applied potential. The initial rate of adsorption of BSA to
the surface can be analyzed by measuring the slope of the accumu-
lated mass over the linear region corresponding with the first min-
utes of BSA adsorption. The rates of adsorption for Fig. 5B are
presented in Fig. 5C. We observe very rapid mass adsorption when
the electrode is positively charged or at 0 V. When the surface and
the molecule are of the same charge, however, the rate of adsorp-
tion is significantly decreased.
Similarly, the apparent adsorption rate constant for BSA to a
negatively charged surface exhibits no distinct transport regimes,
suggesting that suggesting that the electrostatic repulsion between
the protein and the surface is dominating the kinetics of adsorp-
tion. For the neutral and positively charged surfaces, the apparent
adsorption rate constant increases with increasing applied poten-
tial (from 5.2E�5 cm s�1, to 5.5E�5 cm s�1, to 6.0E�5 cm s�1 to
8.2E�5 cm s�1 between neutral, +0.1, +0.3, and +0.5 V, respec-
tively). This finding is consistent with past experiments demon-
strating increasing apparent adsorption rate constants for
negatively charged BSA to a positively charged surface over a
higher range of applied potentials [35].
Fig. 6. Electrochemical behavior of BSA adsorbed on the gold surface. (A) Cyclic voltamm
was adsorbed at a concentration of 0.20 g/L, pH = 5.716, conductivity = 1190.2 lS/cm. 10
of the adsorbed layer of BSA as the cyclic voltammetry progresses, Eoc = 0.174 V, and (
Eoc = 0.178 V.
3.5. Cyclic voltammetry to elucidate conformational changes of BSA at
different applied potentials
We performed cyclic voltammetry (CV) to elucidate the confor-
mational changes of adsorbed BSA under different applied poten-
tials. We report both the cyclic voltammogram of an uncoated
gold electrode and of the gold electrode with adsorbed BSA
(Fig. 6A). We also report E-QCM-D response for the coated and
the uncoated crystal for a voltage scan rate of 20 mV/s for 6 cycles
in Fig. 6B and C, respectively.
The CV profile (Fig. 6A) indicates that BSA does not undergo any
redox processes over the potential range evaluated in this experi-
ment. In the E-QCM-D measurement (Fig. 6B), we observe a small
frequency drop when the electrode is negatively charged. Interest-
ingly, however, we also observe an increase in dissipation for the
negatively charged condition. As the adsorption of Na+ ions should
not influence dissipation, these results suggest that the drop in fre-
quency stems from both increased mass associated with the
adsorption of Na+, as well as changes in the conformation of BSA.
This is further supported by the E-QCM-D result (Fig. 6C) for the
uncoated crystal. The displacement in frequency of 7 Hz is exactly
the same as in Fig. 6B, however, the change in dissipation is roughly
20% of that with the adsorbed BSA layer. This suggests that the
change in mass is largely due to the Na+ adsorption whereas the
change in dissipation is a result of conformational change in BSA.
This hypothesis is supported by previous research on the orien-
tational and conformational changes of albumin protein on
ogram of the adsorbed BSA layer and uncoated gold crystal. Scan rate = 20 mV/s. BSA
mM NaCl solution has pH = 5.317, conductivity = 1213.4 lS/cm, (B) QCM-D response
C) QCM-D response of the uncoated crystal as the cyclic voltammetry progresses,
B. Beykal et al. / Journal of Colloid and Interface Science 460 (2015) 321–328 327
charged surfaces [66], though this past work did not benefit from
the insight provided by dissipation monitoring. Proteins that
undergo simple reorientation (horizontal to vertical or vice versa)
on a surface have both high surface diffusivity and molecular
rigidity. Therefore, BSA’s multiple anchoring sites with metallic
surfaces and its soft structure suggests that it is very unlikely that
BSA will undergo a reorientation on the surface without any
conformational change. Thus, the change in viscoelasticity that
we observe can be attributed to the conformational changes in
the adsorbed protein layer.
This is further understood by recalling earlier discussion about
the conformational forms of BSA [46,51]. Under open circuit poten-
tial and pH = 5.716, BSA molecules adopt their most compact and
rigid conformation. The results here suggest that as the electrode
becomes negatively charged, the electrostatic repulsion between
the negatively charged BSA and the negatively charged surface
cause the protein to adopt a more extended conformation. Under
this condition BSA molecules have a larger hydration sphere,
thereby increasing the mass of the adsorbed layer and making this
layer more viscoelastic. Indeed, we observe an increase in dissipa-
tion of approximately 1.25E�6 (Fig. 6B).
When the voltage is reversed, Na+ ions will desorb from the sur-
face and the BSA will return to its compact conformation. Both con-
tribute to a decrease in adsorbed mass, or an increase in frequency.
Fig. 6B confirms this complete reversal of both frequency and dis-
sipation over multiple cycles.
4. Conclusions
Our work demonstrates that electrostatic interactions play a
key role in total mass adsorption and in rate of adsorption of
BSA, an amphoteric molecule, to charged and uncharged surfaces.
As reported elsewhere, mass adsorption is greater when the sur-
face and the protein are oppositely charged. We also find that
increasing magnitude of the applied potential on the gold electrode
is positively correlated with increasing mass adsorption. On the
other hand, the rate of adsorption depends primarily on the polar-
ity of the applied potential rather than the magnitude of the exter-
nal polarization. Cyclic voltammetry results suggest that changes
in the magnitude and polarity of the electrode induce changes in
the conformation of adsorbed BSA. Under repulsive conditions,
BSA adopts an extended conformation, while under attractive ones,
electrostatic attraction between the surface and BSA causes BSA
molecules to adopt a compact, rigid conformation.
Acknowledgments
We acknowledge the support of the US National Science Foun-
dation under award numbers SEES-1215845 and CBET-1403826,
and the United States-Israel Binational Science Foundation under
award number 2012142.
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	Influence of surface charge on the rate, exte
	1 Introduction
	2 Materials and methods
	2.1 Measuring the zeta potential to determine
	2.2 Measuring adsorption to charged surfaces
	2.3 Preparation of gold-coated quartz crystal
	3 Results and discussion
	3.1 Amphoteric behavior of Bovine Serum Albumin
	3.2 Bovine Serum Albumin adsorption in the ab
	3.3 Effect of protein charge and electrode po
	3.3.1 Negatively charged BSA
	3.3.2 Positively charged BSA
	3.4 Effect of varying the magnitude of applie
	3.5 Cyclic voltammetry to elucidate conformat
	4 Conclusions
	Acknowledgments
	References