Article

pubs.acs.org/Langmuir

Nanoscale Mechano−Electronic Behavior of a Metalloprotein as a

Variable of Metal Content
Tatini Rakshit, Siddhartha Banerjee, Sourav Mishra, and Rupa Mukhopadhyay*
Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India
*
S Supporting Information

ABSTRACT: In this work, we have explored an approach to

finding a correlation between the mechanical response of a
metalloprotein against a range of applied force (by force curve
analysis) and its electrical response under pressure stimulation (by
current sensing atomic force spectroscopy) at the nanoscale. Iron-
storage protein ferritin has been chosen as an experimental model
system because it naturally contains a semiconducting iron core.
This core consists of a large number of iron atoms and is therefore
expected to exert a clear influence on the overall mechanical
response of the protein structure. Four different ferritins
(apoferritin, Fe(III)-ferritins containing ∼750 and ∼1400 iron atoms, and holoferritin containing ∼2600 iron atoms) were
chosen in order to identify any relation between the mechano−electronic behavior of the ferritins and their metal content. We
report the measurement of Young’s modulus values of the ferritin proteins as applicable in a nanoscale environment, for the first
time, and show that these values are directly linked to the iron content of the individual ferritin type. The greater the iron
content, the greater the Young’s modulus and in general the slower the rate of deformation against the application of force. When
compressed, all the four ferritins exhibited increased electronic conductivity. A correlation between the iron content of the
ferritins and the current values observed at certain bias voltages could be made at higher bias values (beyond 0.7 V), but no such
discrimination among the four compressed ferritins could be made at the lower voltages. We propose that only at higher voltages
can the iron atoms that reside deeper inside the core of the ferritins be accessed. The iron atoms that could be situated at the
inner wall of the protein shell appear to make a general contribution to the electronic conductivity of the four ferritin systems.

■ INTRODUCTION
Investigations of the mechanical properties of proteins (e.g.,
elasticity, deformability, compressibility, etc.) are important for
utilizing proteins in different biomaterial applications. So far,
only a few reports on understanding the nanoscale mechanical
properties of globular proteins have been made.1−3 Although a
number of studies on force-induced solid-state electron
transport in globular proteins have been reported4,5 and it
has been largely observed that the application of compressive
force increases the solid-state conductivity of proteins,6,7 little
or no information on a correlation between the elasticity of a
protein and the force-induced conductivity increase has been
offered. In this context, it would be interesting to study a
metalloprotein that contains a large metal core consisting of a
few thousand metal atoms because the presence of a large
number of metal atoms is expected to give rise to distinctively
different results in comparison to the metal-free apoprotein.
Ferritins, the iron-storage proteins, naturally contain a few
thousand iron atoms inside the protein shell. They can
therefore be considered to be an excellent experimental
model system for such a study.
Ferritin is an omnipresent protein because it can be found in
all animals, plants, and bacteria. It has a unique ordered
arrangement of 24 subunits that leads to the formation of a
hollow sphere with an external diameter of approximately 12
nm and an internal diameter of 7 to 8 nm.8 Ferritin can gather
excess free iron in the form of ferrihydrite phosphate in its core
to prevent the harmful accumulation of iron in the body.9 The
core of mammalian holoferritin can contain up to 4500 iron
atoms in the form of ferrihydrite phosphate
[(FeOOH)8(FeOPO3H2)].8 This stored iron is released from
holoferritin by means of the reductive dissolution of the core to
supply the iron required for different cellular processes. Ferritin
is structurally robust and stays in its functional form in an
aqueous environment having a pH of between 4.0 and 9.0 and
well up to a temperature of 80 °C10 and is therefore quite
suitable for on-surface studies under varied experimental
conditions.
Though the mechanical properties of the ferritin proteins
have not been extensively studied, it was reported earlier that
the presence of ferritin nanoparticles in poly(vinyl alcohol)
(PVA) nanofibers showed an enhancement of the elastic
modulus as compared to that of pure PVA nanofibers because
of chemical interactions between ferritin and the PVA matrix.11
Bhattacharyya et al. showed that composite films containing
ferritin-functionalized multiwalled carbon nanotubes
Received: July 3, 2013
Revised: September 11, 2013

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Langmuir
(MWCNTs) were reinforced by the formation of hydrogen
bonds between ferritin-functionalized MWCNTs and the
polymeric films.12
Our present work aims to study the mechanical properties of
the ferritin layers on solid substrates by atomic force
spectroscopy (AFS). Here we used (AFS), a powerful tool to
measure the nanomechanical properties of biological sam-
ples13−19 based on the pushing experiments that can provide
information on the mechanical properties of the samples
through the measurement of approach force curves. Previously,
we reported the pressure-stimulated electrical conductance of
ferritin proteins by current-sensing AFS where we showed that
the electrical conductivity of the ferritin proteins could be
modulated by the application of compressive force.20 Within
this framework, we will try to establish a correlation between
the nanoscale mechanical properties and the pressure-
stimulated electronic response of the ferritin proteins. Four
ferritin systems (i.e., the holoferritin (∼2600 Fe atoms), the
two Fe(III)-ferritins (∼750 and ∼1400 Fe atoms), and the
apoferritin) will be employed so that any role of the metal
content in such correlations can be elicited.
■
MATERIALS AND METHODS
Preparation of Protein Solutions. Equine spleen holoferritin
and apoferritin were obtained from Sigma and used as is. The Fe(III)-
ferritin samples were prepared using a procedure reported by Snow et
al.21 (details in Supporting Information).
Preparation of Substrates. The preparation of the flame-
annealed Au(111) substrate, the NHS-EDC-modified Au(111)
substrate, and the highly oriented pyrolytic graphite (HOPG)
substrate were carried out using standard procedures described in
the Supporting Information.
Preparation of Protein-Modified Surfaces. Protein modifica-
tion of gold substrates was undertaken by immersing a bare Au(111)
or a modified Au(111) substrate into a 20−40 pM ferritin solution at
room temperature (24 ± 1 °C), followed by 12 h of incubation (in the
case of bare gold) or 1 h (in the case of modified gold). The gold piece
was washed with 0.5 mL of filtered tris buffer solution (0.1 M tris and
0.1 M NaCl, pH 8.0) as in the case of holo-/apoferritins or the MOPS
buffer (50 mM MOPS, 0.1 M NaCl, pH 7.5) as in the case of the
Fe(III)-ferritins, followed by washing with 1 mL of Milli-Q water and
drying gently with a stream of nitrogen gas. For AFM imaging in fluid,
the respective buffers (i.e., tris buffer as in the case of holo-/
apoferritins and the MOPS buffer as in the case of the Fe(III)-
ferritins) were applied.
To develop submonolayer deposits of ferritins on an HOPG
surface, 10 μL of a 1.5 nM ferritin solution was deposited onto freshly
cleaved HOPG, kept in a covered Petri dish for 30 min, washed with 1
mL of the tris buffer (0.1 M tris, 0.1 M NaCl, pH 8.0) and 1 mL of
Milli-Q water, and then dried gently under a stream of nitrogen gas.
The AFM images were captured in a buffered medium using tris buffer
(0.1 M tris, 0.1 M NaCl, pH 8.0).
To develop films on the HOPG surface, ferritin solutions of 1.9−2.5
μM concentration were spin-coated at 2800−3000 rpm for 25−30 s
onto a freshly cleaved HOPG substrate. After spin-coating, the sample
was washed with 0.5 mL of the respective buffer solutions followed by
1 mL of Milli-Q water and dried under a weak stream of nitrogen gas.
AFM and AFS Data Acquisition and Analysis. AFM imaging in
intermittent contact mode (AAC mode) and force curve acquisition in
the contact mode were performed using PicoLE AFM equipment
(Agilent Corp., USA) using PicoView 1.12.2 software with a 10 μm ×
10 μm scanner at room temperature (24 ± 1 °C). A single cantilever
(SNL-10, Veeco) was used for all of the experiments described in this
study except for the scratching experiments. The SNL-10 cantilever
was calibrated using the Thermal K program that interfaces with the
imaging software to calculate the spring constant of a cantilever by the
thermal method,22,23 and the calibrated spring constant was found to
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be 0.546 N/m. The probe was cleaned in a UV-ozone cleaner
(Bioforce, Nanosciences) before imaging. The amplitude set point was
85−90% of the free oscillation amplitude, which is 11−13 nm (in
fluid). The scan speed was 1.0−2.0 lines/s. All images presented here
are raw data except for minimum processing limited to first-order to
third-order flattening. The height values were measured as the
difference between the highest point of the cross-sectional diagram and
the average height of the substrate surface. Dimensional analysis was
performed on 50−60 molecules from different images.
To acquire information on the thickness of the ferritin films
generated on bare and modified gold surface, we performed scratching
experiments (details in Supporting Information).
The force curves, consisting of 4000 data points, were obtained by
imposing a maximum applied force of 2.0 nN (for Young’s modulus
measurement). Then the amount of applied force was increased in a
controlled manner starting from 10 to 70 nN with an interval of 10 nN
at each stage using PicoView 1.12.2 software (for deformation study).
The force−distance (F−D) experiments were repeated for ∼500
molecules from different areas as well as from different samples. In all
of the experiments, a 35−45% humidity level was maintained. The
results presented here have been obtained after analyzing 400−500
force curves for each system. To obtain the indentation values, the
photodiode sensitivity was calibrated by obtaining a force curve on
hard bare substrates (a freshly annealed Au(111) surface and a freshly
cleaved HOPG surface) as reference. The HOPG substrate was used
to estimate the effective radius of the AFM tip, which was found to be
7 nm. When a spherical tip scans a HOPG surface containing a step
(step height 0.34 nm) lying along the direction perpendicular to the
scan, if contact between both is established at a distance W, then W =
(2RH − H2)1/2, where R is the tip radius and H is the height of the
step. The tip radius can be determined by using the equation R = (H2
+ W2)/2H (detailed steps in ref 24). After each measurement, the tip
was checked to ensure that no change in the tip integrity (i.e., tip
radius, frequency, and spring constant) took place.
To calculate the Young’s modulus of the proteins, the following
steps were performed. First, the difference between the cantilever
deflections as detected on the hard surface and on the soft protein
samples, which describes the deformation of the protein sample under
the tip load, was calculated.2 The obtained indentation values were
plotted against the force to acquire the indentation versus force curves.
The approaching part of the force curve was considered for calculating
the indentation. Next, the indentation−force curves were fitted to the
Hertz model, which describes the elastic deformation of a soft sample
by a rigid indenter.25 Because ferritin is a globular protein, the same
method as reported earlier by Parra et al.2 was applied. The Hertz
model considering the spherical and conical tip geometry25,26 of the
forms (A) δ = [3(1 − ν2)/(4ER1/2)]2/3F2/3 (for a spherical indenter, eq
1) and (B) δ = [π(1 − ν2)/(2E tan α)]1/2F1/2 (for a conical indenter,
eq 2) was used, where ν is the Poisson ratio of the sample, which is
assumed to be 1/3 as in studies reported by Radmacher et al.1 and
Parra et al.2 who used a silicon nitride lever similar to the SNL-10
cantilever used in the present study. Here, δ, F, E, R, and α are the
indentation, force, Young’s modulus of the sample, radius of the tip,
and half-opening angle of the conical tip, respectively.
Current (I)−Voltage (V) Acquisition by Current-Sensing AFS.
The I−V measurements on the spin-coated ferritin films were made
using CSAFS in contact mode where the electrical characteristics were
recorded independent of force feedback. In CSAFS experiments, Ti/Pt
coated cantilevers (Micromasch, Estonia) having a spring constant of 2
N/m, a tip radius of ∼35 nm, a length of 110 μm, and a width of 40
μm were used. The conductive probe was initially engaged with the
sample at minimal force (in force feedback). The I−V sweeps were
made between ±1.5 at 1 V/s (given as a tip bias with respect to the
substrate), and the resulting current responses were recorded. The I−
V experiments were repeated for ∼100 different locations from
different areas as well as from different samples. Each I−V spectrum
acquired on a protein molecule was an average over five sweeps.
During each measurement, the force set point was calibrated and
adjusted as required. To calculate the absolute force at a particular
point, the force−distance curves were recorded to relate the force set
Langmuir
point to the cantilever deflection. All CSAFS measurements were
performed under ambient conditions where the temperature and
humidity were maintained at 24 ± 1 °C and 35−45%, respectively.
The current−voltage measurements were not made in buffer solution
in order to avoid extraneous influences of buffer ions on the
measurements. In the case of a storage metalloprotein such as ferritin,
such a non-physiological condition may be less important than for
small functional enzymes or electron-transfer proteins. Also, because
the water molecules would be present on the protein exterior, which is
the same for all four ferritin systems, this factor could be a common
source of error for all of the ferritin samples that we studied in this
work. Thus, the presence of moisture would not alter the comparative
picture that is elicited from the present study.
■
RESULTS AND DISCUSSION
In the present study, the relationship between the mechanical
response of ferritins with four different iron contents
(apoferritin, ∼750 and ∼1400 iron-containing Fe(III)-ferritins,
and ∼2600 iron-containing holoferritin) against an applied
force within 2 and/or 10−70 nN (by force curve analysis) and
the electrical response of these ferritin systems under pressure
stimulation across 17−66 nN (by CSAFS measurements) has
been explored. All of the measurements were performed on
ferritin film structures to minimize the influence of surface
defects on AFM/AFS observations. To determine the role of
the substrate in the elastic response of the ferritins, the force
curve measurements were made on bare Au(111), 3-MPA-
modified Au(111), and freshly cleaved HOPG substrates. In
this study, all of the AFM/AFS experiments were carried out in
a buffered medium.
Formation of Ferritin Film Structures on a Gold
Surface. Figure 1 shows the characteristic in situ AFM
Figure 1. AFM topographic images for (A) the 3-MPA-NHS-EDC-
modified Au(111) surface, (B) apoferritin, (C) holoferritin, (D)
Fe(III)-ferritin (∼1400), and (E) Fe(III)-ferritin (∼750) films on the
modified Au(111) surface, taken under buffered conditions. z ranges
for (A) 0−4.1, (B) 0−4.5, (C) 0−4.0, (D) 0−6.1, and (E) 0−4.3 nm;
the scale bar is 150 nm.
topographic images of the modified Au(111) substrate and the
ferritin films on the modified Au(111) substrate. Although the
protein molecular contours could be well resolved on the
modified Au(111) substrate, the individual molecules could
barely be visualized, and streaky features were routinely
observed on the bare Au(111) surface (Figure S3 in the
Supporting Information), indicating better anchoring of the
proteins on the modified gold surface than on the bare gold
surface. Evidently, the NHS-EDC-assisted covalent amide
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linkage formation between the amine groups of the surface-
exposed lysine residues of ferritin and the carboxylic ends of the
3-MPA molecules,27 which are immobilized on the gold surface
via strong gold−thiol interactions,28 appeared to be a better
strategy than direct immobilization of the ferritin molecules via
gold−sulfur interactions.29 Recently, Kim et al. experimentally
verified the immobilization of ferritin molecules on gold
surfaces via gold−sulfur interactions.30 The ferritin films that
we used are likely to be monolayers, as apparent from the
results of the contact mode scratching experiments on the films
prepared on both modified and bare gold substrates. The
observed shapes of ferritin molecules were generally spherical,
which conforms well to the respective X-ray structures.31,32 The
width values as obtained from intermittent contact AFM
imaging are generally found to be less informative as a result of
a relatively large tip radius (∼7 nm in the present case) and the
tip geometry-induced convolution effects.33 Therefore, the
AFM width values were not measured although we verified that
the molecules were similarly sized, which indicates that no
major aggregation of the ferritins took place upon adsorption.
The measured averaged height values for apoferritin and
holoferritin molecules, as obtained from the images of the
protein films on the bare Au(111) surface, were found to be 4.5
± 1.1 and 5.2 ± 0.7 nm, respectively. The discrepancy between
the theoretical value (∼12 nm)8 and the experimentally
determined values can be explained in terms of tip-induced
compression of the soft protein exterior of the ferritin
molecules, which are directly adsorbed on the hard solid-gold
surface. However, when the protein molecules were not directly
exposed to the gold surface but were adsorbed onto a layer of
3-MPA, increased height values for both apoferritin and
holoferritin molecules (i.e., 8.5 ± 0.9 and 9.0 ± 1.0 nm,
respectively) were observed, indicating less tip-induced
compression of the ferritin proteins on the modified surface.
This observation of increased height values of the proteins on
the modified gold surface could also indicate less adsorption-
induced deformation/flattening, which is typically associated
with substrate−molecule non-specific electrostatic interactions.
Measurement of Young’s Modulus of the Ferritin
Proteins. To quantify the mechanical properties of the
ferritins, force curve (force vs distance) experiments were
executed using a cantilever of calibrated force constant value
0.546 N/m. In Figure 2A, an overlay of the averaged approach
force curves obtained for bare Au(111), holoferritin, and
apoferritin on bare Au(111) is shown. Noticeably different
cantilever deflection behavior in these three cases could be
observed, indicating clearly different surface hardness proper-
ties. Figure 2B shows the overlay of averaged approach force
curves for bare Au(111), modified Au(111), holoferritin, and
apoferritin on a modified Au(111) surface. The observation
that the distance (or piezo displacement) is shorter (for the
same value of the cantilever deflection) for the modified gold
substrate than for the bare gold substrate indicates that the
indentation of the protein layer by the cantilever is greater for
the protein films prepared on the bare gold substrate. The
overlay of the averaged approach force curves for holoferritin,
the two Fe(III)-ferritin proteins, and apoferritin on the
modified gold surface is presented in Figure 2C. Clear
differences in the cantilever deflection behavior among the
four differently iron-loaded ferritins could be observed.
Interestingly, it was found that for a fixed distance the
cantilever deflection value for the four ferritin systems was
directly proportional to the iron content/molecule. An
Langmuir Article

Figure 2. Overlay of averaged force curves for (A) bare Au(111), holoferritin, and apoferritin on bare Au(111), (B) bare Au(111), modified
Au(111), holoferritin, and apoferritin on modified Au(111), and (C) holoferritin, Fe(III)-ferritins (∼750 and ∼1400), and apoferritin on modified
Au(111). Indentation vs force curves for (D) apoferritin and holoferritin on bare Au(111) and (E) apoferritin, Fe(III)-ferritins (∼750 and ∼1400),
and holoferritin on modified Au(111). Solid lines indicate the fitting of the respective curves following the Hertz model for a spherical indenter, (F)
plots of averaged values of the Young’s modulus for four ferritin systems vs no. of iron atoms loaded per ferritin molecule.

increasingly greater extent of indentation for apoferritin than
for holoferritin as the force value was increased could be
observed on both the bare gold and the modified gold surfaces
(Figure 2D, E). This probably reflects the fact that the presence
or absence of the iron core makes a clearly detectable difference
in the indentation profiles. A comparison among the
indentation versus force profiles for the four ferritin systems
acquired on the modified Au(111) surface (Figure 2E) suggests
that the extent of their indentations for the same amount of
force applied follows an order that is the inverse of the order of
the metal content (i.e., holoferritin (∼2600) > Fe(III)-ferritin
(∼1400) > Fe(III)-ferritin (∼750) > apoferritin).
The indentation versus force profiles were fitted with the
Hertz model, where the fitting could be better performed with
eq 1 (spherical geometry) (Figure 2D, E) than with eq 2
(conical geometry) (Figure S4 in Supporting Information).
This indicates that the spherical tip geometry is more adequate
for explaining our experimental data. The indentation values
measured for both apoferritin and holoferritin on a modified
surface were smaller than on the bare Au(111) surface, which
was reflected in the greater Young’s modulus values obtained in
the case of the modified Au(111) surface (Table 1) compared
to the values obtained (0.25 ± 0.1 and 0.43 ± 0.1 GPa for
apoferritin and holoferritin, respectively) on the bare Au(111)
surface. To calculate the Young’s modulus for the 3-MPA-
NHS-EDC layer, because under similar loads, the NHS-EDC
and protein layers are compressed to similar extents (Figure
2B), which implies that the calculated Young’s modulus values
Table 1. Averaged Values of the Young’s Modulus of the
Ferritin Proteins in Case of the Molecules Adsorbed onto
Modified Au(111) and Assuming Spherical Tip Geometry
ferritins Young’s modulus (GPa)
holoferritin 0.68 ± 0.2
Fe(III)-ferritin (∼1400) 0.52 ± 0.2
Fe(III)-ferritin (∼750) 0.42 ± 0.1
apoferritin 0.35 ± 0.1
D dx.doi.org/10.1021/la402522m | Langmuir XXXX, XXX, XXX−XXX
have a significant contribution from the compression of the
NHS-EDC layer (even though the relative trend would not
change), we tried to fit the force−indentation profile for this
layer with the Hertz model, but the data could not be well-fitted
with the equation. The Young’s modulus values of apoferritin
and holoferritin were increased by 41 and 57%, respectively, on
the modified gold surface. It is worth mentioning here that the
Young’s modulus value of holoferritin was 1.72 times higher
than that for apoferritin on the bare Au(111) surface whereas it
was 1.91 times higher in case of the modified Au(111) surface.
These observations mean that both ferritins were more rigid
(or less compressible) on the modified surface, and their elastic
behavior could be better discriminated on the modified surface.
Figure 2F shows the plots of averaged values of the Young’s
modulus of four ferritin systems versus the number of iron
atoms loaded per ferritin molecule, which reveals that the
extent of metal loading has a direct influence on the elastic
behavior of the ferritin systems. Few previous reports on the
Young’s modulus of the globular proteins by AFM,1−3
considering spherical tip geometry, are found to match well
with our derived Young’s modulus values. The Young’s
modulus values that we obtained in the case of conical
geometry were expectedly quite different (Table S1 in
Supporting Information).
Mechanical Response of the Ferritin Systems and Its
Relation to the Proteins’ Electronic Response under
Compressive Force Loads. To identify a relationship
between the mechanical response of ferritins with different
iron contents under various force loads and their electron
transport behavior with respect to the applied force at the
nanoscale, we first measured the indentation values for
apoferritin and holoferritin films along with the two Fe(III)-
ferritins (∼750 and ∼1400) against different applied forces
starting from 10 to 70 nN at 10 nN intervals on a modified
Au(111) surface (Figure 3A−G). In general, each of the curves
obtained in these set of experiments was nonlinear. With
increasing applied forces, the indentation values for all four
proteins increased monotonously. From the plots of the highest
Langmuir
Figure 3. (A−G) Averaged deformation (nm) vs force (nN) curves for
apoferritin, Fe(III)-ferritins (∼750 and ∼1400), and holoferritin on
the modified Au(111) surface at increasing forces applied starting from
10 to 70 nN. (H) Plots for the highest deformation (nm) vs the
respective force value (nN) (with mean deviations shown) for
apoferritin, Fe(III)-ferritins (∼750 and ∼1400), and holoferritin on a
modified Au(111) surface.
deformation values for the different forces applied (Figure 3H),
more or less clear differences could be observed among the four
protein systems, although only for the high force values (i.e., 40
nN and above). However, differences observed between
apoferritin and Fe(III)-ferritin (∼750) and between holoferritin
and Fe(III)-ferritin (∼1400) were rather small. It is worth
mentioning here that the deformation induced on protein films
under the highest load (i.e., 70 nN) reached values that were
higher than the film thickness. This could be related to the non-
linearity of cantilever deflection for high deflection values34 and
probably also having contributions from the compression of the
underlying 3-MPA layer. From Figure 3A−G, it is evident that
the indentation values measured for apoferritin were always
greater in comparison to those for holoferritin (i.e., iron-loaded
holoferritin molecules were more resistant to the applied force-
induced indentation than were empty apoferritin molecules).
From the plots of the rate of deformation with increasing
applied force load on each system (Figure 4), it was found that
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Figure 4. Plots of the rate of deformation (nm/nN) values against the
applied force loads (nN) for apoferritin, Fe(III)-ferritins (∼750 and
∼1400), and holoferritin.
the rate of deformation for apoferritin increased up to 40 nN of
applied force, beyond which it decreased slightly. In contrast, in
the case of holoferritin, the rate of deformation decreased from
the beginning (i.e., from 10 to 50 nN), after which it became
almost constant and increased a little. Similarly, in the case of
the Fe(III)-ferritin samples, the rate of deformation decreased
from the beginning up to 50 nN (in the case of Fe(III)-ferritin
(∼1400)) or 60 nN (in case of Fe(III)-ferritin (∼750)).
Beyond these points, the rate of deformation increased for both
Fe(III)-ferritins. From these data, we can infer that apoferritin
behaves drastically different than the other three ferritin
systems, most likely as a result of the major structural difference
due to the absence of the iron core. The initial increase in the
rate of deformation in the case of apoferritin could be due to
the ease with which the AFM probe could indent the protein
molecule, which is devoid of the iron core. After a certain
threshold value of the atomic packing density was reached,
further compression became gradually ineffective and the rate
of deformation was reduced. In the case of the iron-containing
ferritins, probably the presence of the hard core introduced a
general rigidity into whole protein molecules, which is reflected
in their tendency to withstand deformation. However, a certain
structural change (or loss) is indicated after the application of a
large amount of force (beyond 50−60 nN), which allowed
deformation to occur more easily, leading to an increase in the
rate of deformation.
Although in the case of naturally occurring holoferritin, a
dense core structure is known to form and a number of reports
have been made on the core structure (e.g., that it could be a
heterogeneous solid with a dense nucleus and could spread out
like tree roots or the rock piles35) and the core formation
mechanism (i.e., a nucleation-driven process36,37), in the
present context we have no clear experimental evidence that
dense iron core structures were formed for Fe(III)-ferritins
containing ∼750 and ∼1400 iron atoms. However, because the
rate of deformation versus force plots (Figure 4) for these two
proteins are more like the plot for holoferritin, we concluded
that the protein behavior was related to the iron core and not
the protein part alone (as in the case of apoferritin). The
observation that the plot for Fe(III)-ferritin containing ∼1400
iron atoms is more similar to the plot for holoferritin than to
Fe(III)-ferritin containing ∼750 iron atoms probably indicates
that the core structures formed in the two Fe(III)-ferritins are
different in nature.
The electrical conductance experiments were performed on
HOPG because electrical contacts with ferritin molecules
Langmuir
adsorbed on the modified Au(111) surface could not be made
by a CSAFS tip, and most often I−V responses representing
direct contact with a conductive substrate or dielectric
breakdown type of behavior were observed. Because it was
shown earlier that the I−V responses of ferritins obtained from
STS (on the bare Au(111) surface) and from CSAFS (on
HOPG surface) experiments were quite similar,20 in the present
framework, the correlation between electrical and mechanical
properties of ferritins is expected to be realistic. However, we
still checked for the deformation behavior of ferritins adsorbed
on the HOPG substrate by first forming a sub-monolayer
deposit of ferritins onto HOPG, followed by AFS measure-
ments. We worked on the sub-monolayer deposit (Figure 5) so
Figure 5. AFM topographic image (3D view) showing the
characteristic sub-monolayer deposit of ferritin proteins on a bare
HOPG substrate obtained under buffered condition.
that we could identify if there was a significant effect of the
substrate present in the observed indentation behavior. The
height of the ferritin molecule adsorbed on a bare HOPG
substrate as a submonolayer deposit under buffered condition
was found to be 7−9 nm. Lesser cantilever deflection and a
greater extent of deformation could be observed in case of the
apo protein compared to the holo form (Figure 6A, B), as also
observed earlier in case of the modified gold or bare gold
substrates (Figure 2). Only small differences could be detected
in the cantilever deflection profiles and the indentation profiles
of both apoferritin and holoferritin when a comparison was
made between the two cases of HOPG substrate and the
modified Au(111) substrate (Figure 6C−F). As observed in the
case of modified Au(111) earlier (Figure 2E), better fitting of
the indentation profiles could be obtained using the equation
for spherical tip geometry. The Young’s modulus values
obtained for the HOPG substrate (0.60 ± 0.2 and 0.31 ± 0.1
GPa for holoferritin and apoferritin, respectively) were found to
be comparable to those obtained for modified Au(111) (Table
1), indicating that there was no major role of the substrate as
long as the comparison was made between modified Au(111)
and HOPG substrates. However, the behavior was drastically
different for bare gold, which is probably to be expected
because HOPG is a fundamentally different substrate compared
to gold, having considerably less surface energy than that of a
gold surface.38,39
For the I−V experiments, the estimated contact area between
the CSAFS tip and the protein film was found to be 630 nm2
considering the tip-induced deformation of the ferritin shell to
be 3 nm because the average height for single ferritin molecules
considering apoferritin as well as the metal-loaded ferritins was
F dx.doi.org/10.1021/la402522m | Langmuir XXXX, XXX, XXX−XXX
Article
Figure 6. (A) Overlay of averaged force curves for HOPG,
holoferritin, and apoferritin on HOPG. (B) Indentation vs force
curves for apoferritin and holoferritin on HOPG. (C) Overlay of
averaged force curves and (D) indentation vs force curves for
apoferritin on bare Au(111), HOPG, and modified Au(111). (E)
Overlay of averaged force curves and (F) indentation vs force curves
for holoferritin on bare Au(111), HOPG, and modified Au(111). (B,
D, F) Solid lines indicate the fitting of the respective curves following
the Hertz model for a spherical indenter.
∼9 nm, which is 3 nm less than ferritin’s crystallographic
diameter of 12 nm.8 This contact area could contain a
maximum of five to six molecules because the area that each
ferritin molecule would occupy is 113 nm2. The present I−V
measurements can therefore be considered to be close to a
molecularly resolved measurement.
In the case of pressure-modulated electronic conduction via
apoferritin, it was shown earlier that the current values
increased more drastically beyond a certain force value,20 in
contrast to holoferritin, which exhibited a more steady increase
in current with incremental changes (Figure 2 in ref 20). For
apoferritin, the junction resistance (R) remained unchanged at
60−65 GΩ for the force range of 20−50 nN. The R value
gradually decreased to 45 GΩ for forces >50−60 nN, and it
decreased to 20 GΩ for the highest force value of 66 nN,
indicating an abrupt increase in current as the force was
increased to >60 nN. This could be an indication of the
pressure-induced partial collapse of the central cavity of
apoferritin. The R values for holoferritin and the Fe(III)-
ferritins at particular forces were comparable and were within a
narrow range: the initial R values at 16−20 nN were 22−23
GΩ, and at 65−66 nN, they were about 2−3.5 GΩ. The iron-
containing ferritins therefore underwent a smaller decrease in
resistance values with increased force application, in contrast to
apoferritin (Table 2).
Langmuir Article

Table 2. Averaged Resistance Values (R) Obtained for Four iron-containing ferritin systems at higher applied force loads.
Different Ferritin Systems by Current Sensing AFS The apoferritin system behaved clearly differently because the
averaged initial and final band gap energies were found to be
difference
ferritins R(GΩ) at 16−20 nN R(GΩ) at 65−66 nN (GΩ) 1.48 and 1.23 eV (Figure 7). An overlay of the averaged I−V
apoferritin 60 20 40
Fe-ferritin 23 2.2 20.8
(∼750)
Fe-ferritin 23 3.1 19.9
(∼1400)
holoferritin 22 3.6 18.4

The reversibility of the I−V profiles as a function of applied

force was checked, and it was revealed that the I−V behavior
was more or less reversible in the lower force regime of 20−35
nN for apoferritin and holoferritin. In this force regime, if the
applied force was increased (by an increment of 5−7 nN at
each step), then the current value gradually increased, and if the
applied force load was decreased in the same manner (by a
decrement of 5−7 nN at each step), then the current decreased
almost in the same proportion. However, in the higher force
regime (>60 nN), generally the current decreased with a
decrease in the applied force load, but not in the same
proportion compared to the increments observed during the
load increase steps. This probably indicates that at a critical
pressure the protein molecules undergo a certain degree of loss Figure 7. Overlay of averaged I−V responses for apoferritin, Fe(III)-
of structure, though not totally unfolded. Previous conductance ferritins (∼750 and ∼1400), and holoferritin obtained for (A) the
studies also suggested that the compressed protein molecules minimum applied force values (16−20 nN) and (B) the maximum
fall short to revert to their original vertical dimensions on applied force values (65−66 nN). Overlay of averaged conductance
curves of the four ferritin systems for (C) the minimum applied force
reduction of applied force, which means that a certain degree of
values (16−20 nN) and (D) the maximum applied force values (65−
loss of structure or irreversible protein deformation can take 66 nN). Bias range ±1.5 V.
place upon compression.7 No clear reversibility in the behavior
of the two Fe(III)-ferritins could be observed, which probably
indicates certain structural differences in comparison to the responses and conductance curves of the four different ferritin
naturally occurring apoferritin and holoferritin. It is likely that a systems for minimum (16−20 nN) and maximum (65−66 nN)
functionally effective protein fold could not be attained in these applied forces is shown in Figure 7.
two “unnatural” cases probably as a result of the fact that a To determine whether the effect of a high force load could
certain threshold number of iron atoms were not present inside reveal any characteristic trend in the conductivities of the
these two types of ferritin molecules. ferritins as a function of metal content, the current values were
We have shown in the previous sections that with increasing measured for the four ferritin systems at different bias voltages.
applied loads (until 50−60 nN) the indentation values did not For example, for the 1.1 V bias, the current values for
increase appreciably in case of the iron-containing ferritins, apoferritin were 0.26 nA for the minimum force and 1.16 nA
which clearly revealed the fact that the presence of a hard metal for the maximum force applied. In the case of the Fe(III)-
core inside the protein structure played a key role in resisting ferritins and holoferritin, the current values were 0.87−0.94 nA
the force-induced deformation. In the case of pressure- for the minimum force situation (Table S2 in Supporting
modulated electronic conduction through holoferritin, a stable Information), and the current values were 2.23, 2.45, and 2.96
incremental increase in current with increasing applied force nA, respectively, for the maximum force applied (Table 3).
loads was observed (Figure S5 in Supporting Information). The Though the current values for three metal-loaded ferritin
electronic conduction through the Fe(III)-ferritins at different systems were comparable to each other for the minimum force
applied force loads was measured, and the band gap values of range (16−20 nN), the currents measured at 65−66 nN could
these systems were found to be comparable to those of be well-discriminated and they followed an order that is the
holoferritin within the 17−66 nN force range (Figure S5 in the
Supporting Information). For the Fe(III)-ferritin (∼750) and Table 3. Averaged Values of Current Obtained for Four
Fe(III)-ferritin (∼1400) systems, the averaged initial (for 16− Different Ferritin Systems by Current Sensing AFS for the
20 nN force applied) band gap energies were 1.05 and 1.11 eV, Maximum Forces (65−66 nN) Applied
respectively, and the final (for 65−66 nN force applied) band
gap energies were 0.77 and 0.61 eV, respectively. These values current current current current
values (nA) values (nA) values (nA) values (nA)
are found to be comparable to the corresponding values ferritins at 0.7 V at 0.9 V at 1.1 V at 1.3 V
obtained for holoferritin that contains 2660 Fe atoms (1.08 eV apoferritin 0.15 0.44 1.16 3.26
for the low force situation and 0.81 eV for the high force Fe(III)-ferritin 0.46 1.02 2.23 4.33
situation). Although the band gap values at 16−20 nN were (∼750)
very similar, the difference in magnitude of the band gaps Fe(III)-ferritin 0.48 1.11 2.45 5.37
increased for the 65−66 nN force applied, indicating better (∼1400)
discrimination of the electrical conductivities of these three holoferritin 0.66 1.45 2.96 5.43

G dx.doi.org/10.1021/la402522m | Langmuir XXXX, XXX, XXX−XXX

Langmuir
same as that for the metal content. A similar observation of a
direct correlation between the current values at high force
situation and the metal content could be made for other bias
voltages of 0.7, 0.9, and 1.3 V as well (Table 3).
The slopes of the I−V curves (considering the positive side
only) were greater for the high-force situation compared to
those for the low-force situation for all the four proteins,
irrespective of the metal content (Table S3 in the Supporting
Information). This observation correlates well with the fact that
for the high-force situation the atomic packing density of the
protein shell part, which is common in all four cases, becomes
high enough to allow a rapid increase in current for the smallest
change in potential. That the compression-induced increase in
current has an active contribution from the compression of the
protein shell of the ferritin proteins is also evident from the
observation that the slope values in the case of the metal-free
apoferritin were the maximum among the four ferritin systems.
We conclude that the extent of metal loading is probably not
the sole determining factor in pressure-modulated electronic
conduction through the ferritins. Had it been so, the
conductance of holoferritin (∼2600 iron atoms) at any applied
force load would have been greater than that of the Fe(III)-
ferritin containing ∼750 iron atoms. In the present study, we
have observed that the rates of deformation for both Fe(III)-
ferritin systems (∼750 and ∼1400) were comparable to those
for holoferritin (Figure 4). This could imply that with the
addition of metal atoms inside the apoferritin cavity the overall
protein structure generally became more rigid, irrespective of
the total number of metal atoms. This could happen if the
metal atoms were predominantly aligned along the inner walls
of the protein shell, rather than forming condensed aggregates
alone in a localized manner. An earlier study of the Co(III)-
ferritin system, which reports the formation of hollow cobalt
nanoparticles consisting of approximately 1000−2000 cobalt
atoms inside the apoferritin cavity, partially supports this
proposition.40 It is apparent that the contribution from the
metal part of these proteins in their force-induced conductance
profiles arises from the metal atoms that reside along the inner
wall of the protein shell, at least for the lower force ranges.
Only when a large amount of force is applied and the protein is
compressed enough could the metal atoms that reside deeper
inside the core be accessed, making the metal content a
determining factor of the force-induced conductance profile.
■
CONCLUSIONS
In the present study, the nanoscale mechano−electronic
behavior of four ferritin systems with differently loaded iron
atoms (apoferritin, Fe(III)-ferritins (∼750 and ∼1400 iron
atoms), and holoferritin (∼2600 iron atoms)) has been
investigated against a range of force applied by CSAFS. We
show from the measured Young’s modulus values that the
amount of iron loaded per ferritin molecule can be directly
linked to ferritin’s mechanical response to compressive force.
Although in general the force-induced increase in current
appears to have a significant contribution from the protein shell
part, the contribution of the metal content in observed force-
induced conductance profiles becomes evident when the
protein molecule is compressed to a significant extent (here
above 65 nN). In the future, it would be interesting to study
other ferritin-like proteins and/or nonprotein systems having a
metal core surrounded by a soft polymeric envelope in order to
assess whether our propositions are generally valid.
H dx.doi.org/10.1021/la402522m | Langmuir XXXX, XXX, XXX−XXX
■
Article
ASSOCIATED CONTENT
*
S Supporting Information
Preparative methods, data acquisition/analysis details, UV−vis
spectra of ferritin samples, TEM images, AFM images on a bare
gold(111) surface, indentation versus force curves with fitting
to a conical indenter equation, Young’s modulus values
considering a conical indenter, AFM images of ferritin films
on HOPG, respective I−V curves across a range of tip−sample
forces, current values at low force, and slope values for low and
high force situations. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: bcrm@iacs.res.in.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We gratefully acknowledge the financial support from DBT,
Government of India (grant no. BT/PR-11765/MED/32/107/
2009), research fellowships to T.R., S.B., and S.M. from the
Indian Association for the Cultivation of Science, Kolkata, and
the Council of Scientific and Industrial Research, Government
of India, respectively. We also thank Dr. S. Ray of Department
of Material Science, IACS, Kolkata, for ICP measurements.
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