Article

pubs.acs.org/Langmuir

Characterization of Polydopamine Thin Films Deposited at Short

Times by Autoxidation of Dopamine
Rebecca A. Zangmeister,* Todd A. Morris,† and Michael J. Tarlov
Biomolecular Measurement Division, Material Measurement Laboratory, National Institute of Standards and Technology, 100 Bureau
Drive, Gaithersburg, Maryland 20899, United States
*
S Supporting Information
Downloaded by UNIV OF ALABAMA at 07:12:11:275 on May 30, 2019
from https://pubs.acs.org/doi/10.1021/la400587j.

ABSTRACT: Current interest in melanin films derived from the autoxidation of dopamine stems from their use as a universal
adhesion layer. Here we report chemical and physical characterization of polydopamine films deposited on gold surfaces from
stirred basic solutions at times ranging from 2 to 60 min, with a focus on times ≤10 min. Data from Fourier transform infrared
(FTIR), X-ray photoelectron spectroscopy (XPS), and electrochemical methods suggest the presence of starting (dopamine) and
intermediate (C=N-containing tautomers of quinone and indole) species in the polydopamine films at all deposition times. A
uniform overlayer analysis of the XPS data indicates that film thickness increased linearly at short deposition times of ≤10 min.
At deposition times ≥10 min, the films appeared largely continuous with surface roughness ≈ ≤ 2 nm, as determined by atomic
force microscopy (AFM). Pinhole-free films, as determined by anionic redox probe measurements, required deposition times of
60 min or greater.

■ INTRODUCTION
Melanin polymers (also known as eumelanin, polydopamine,
dopamine-melanin, Pdop, and melanoid pigments) have been
studied for over a century.1 Early interest in these naturally
occurring dark-colored polymers stemmed from their role in
the coloration of human skin and neurological diseases such as
Parkinson’s disease.1−3 In the Raper−Mason reaction scheme,
developed midcentury, the formation of melanin polymers
starts with the enzymatic conversion of tyrosine to L-DOPA [3-
(3,4-dihydroxyphenyl)-1-alanine] by tyrosinase. Reacting
through several steps, the L-DOPA is eventually converted to
5,6-dihydroxyindole, which is thought to be the key monomer
involved in polymer formation.4,5 Since the Raper−Mason
model was first proposed, numerous studies characterizing
melanin films derived from both natural sources (e.g., sepia
cuttlefish) and synthetically produced melanins have been
reported. Synthetic melanins have been produced from
different starting materials including L-DOPA, dopamine, 5,6-
dihydroxyindole (DHI), and 5,6-dihydroxyindole-2-carboxylic
acid by enzymatic or by oxidative polymerization.6−12 These
studies, along with others who used spectroscopic7,13−17 and
electrochemical18−22 measurements, have largely confirmed the
Raper−Mason model and identified new intermediate species.
The most recent structural investigations of synthetic
eumelanin polymers suggest that the polymer is composed of
This article not subject to U.S. Copyright.
Published 2013 by the American Chemical
Society
aggregates of oligomers resulting from the polymerization of
DHI.11,12
Interest in the studies of melanin polymers, synthetically
produced by the autoxidation of dopamine, are motivated by
the use of this material as a universal adhesion layer.23 It was
reported by Lee et al.23−27 that a dopamine-derived melanin
polymer (coined polydopamine) formed in solution can adsorb
to many surfaces, where it can function as an adhesion layer for
the immobilization of biological molecules and mercapto-
functionalized self-assembled monolayers. Further studies have
shown that polydopamine can been used as an adhesive layer
on a variety of surfaces24,28,29 and nanoparticles,30 in a modified
form as nonadhesive layers,31 as a bio-optoelectronic material,8
and as a fouling-resistant layer on water purification
membranes.32,33 Additional studies have examined deposition
times and solution conditions in detail to better understand and
control the film formation process.21,34−37 We previously
reported the use of polydopamine films to attach lectins to gold
surfaces for surface plasmon resonance (SPR) studies of
glycan−lectin interactions.38 These polydopamine films were
deposited from solution according to the method published by
Lee et al.25 and are generated through the autoxidation9 of
Received: February 13, 2013
Revised: May 17, 2013
Published: June 10, 2013
8619 dx.doi.org/10.1021/la400587j | Langmuir 2013, 29, 8619−8628
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Figure 1. (a) Contemporary reaction mechanism of polydopamine from dopamine,11,12,23,29,36,40,50 showing intermediate species. (b) Structures of
intermediate species 5,6-dihydroxyindole and 5,6-indolequinone and their tautomers8,39 that contain C=N functional groups.
dopamine hydrochloride in an aqueous solution kept at basic
pH (8.5). This work prompted us to seek out the minimum
deposition time for the formation of a continuous film of
polydopamine that has physicochemical properties similar to
“bulk” films deposited under the same conditions but at much
longer times.
The reaction mechanism of the formation of synthetic
melanin from the autoxidation of dopamine (polydopamine)
has similarities to the Raper−Mason reaction mechanism for
naturally derived melanin formed from tyrosine. A representa-
tion of a contemporary mechanism for polydopamine is
presented in Figure 1. It is commonly accepted that reaction
intermediate indole species, 5,6-hydroxyindole and 5,6-
indolequinone, are formed and that they react to form the
resulting dopamine-based melanin material polydopamine.
Several different tautomers of these molecules can exist (Figure
1b) and have been suggested to be present in melanin.39 The
precise structure of polydopamine is more difficult to determine
due to the heterogeneity and insolubility of the material. There
is evidence that polydopamine is composed of oligomers of
covalently bound dimers and higher oligomers of 5,6-
hydroxyindole and 5,6-indolequinone held together by charge
transfer, π-stacking, and hydrogen bonding.8,11,12,40
Most characterization studies of melanin polymers, including
melanin derived from dopamine, have been done on purified
bulk melanin polymer material derived from natural sources or
from the enzymatic or autoxidation of known precursor
molecules.1,2,7,9,13−17,41,42 Bernsmann et al.35 published an
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extensive (XPS, AFM, ellipsometry, QCM, UV−vis, and
electrochemical) analysis of melanin-dopamine (synonymous
with polydopamine) thin f ilms, not bulk melanin polymer,
deposited in situ with a minimum deposition time of 15 min.
They reported that thick films of melanin-dopamine can be
achieved by using multiple dip cycles in unstirred fresh
dopamine solutions, based on the observation that nonoxidized
dopamine is necessary for deposition on solid surfaces.34−37 In
a recent study, Bernsmann et al.21 showed that the permeability
and permselectivity of dopamine-melanin films can be tuned
according to the method used for deposition of the material
(i.e., oxidative vs electrochemical polymerization). Kim et al.43
have also recently shown that deposition from a dopamine
solution under a pure oxygen environment results in an
increase in the reaction kinetics of alkaline dopamine oxidation
and a homogeneous, smooth polydopamine film.
None of these studies has investigated the physicochemical
characterization of polydopamine films at short deposition
times, ≤10 min. Our aim was to focus on short deposition
times in order to determine the minimum deposition time for
the formation of a continuous film of polydopamine that has
physicochemical properties similar to “bulk” films deposited
under the same conditions but at much longer times. Here, we
present an extensive physicochemical characterization of
polydopamine films deposited on gold surfaces for short
times (2−10 min) from a stirred basic solution of dopamine.
Our work complements the previous work (referred to in the
preceding paragraph) and offers new characterization data of
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polydopamine films from short deposition times. Stirring, as
well as vertical sample orientation, has been noted as a way of
avoiding the deposition of micrometer-sized particles of
polydopamine on surfaces.23 We hypothesized that using
vertical sample orientation and very short deposition times
would result in the deposition of thin, reproducible, and
possibly continuous films of polydopamine. Identification of the
minimum deposition time necessary to form a continuous film
of polydopamine is an aim of our work and is valuable
information for using polydopamine as an adhesion layer in
applications involving scanning probe measurements, reflec-
tometry, surface-enhanced Raman, or SPR.
This is the first report of physicochemical characterization of
polydopamine deposited from a basic stirred solution at times
≤10 min. Atomic force microscopy (AFM), electrochemical
analysis, Fourier transform infrared (FTIR) spectroscopy, and
X-ray photoelectron spectroscopy (XPS) were used in
characterizing films. AFM was used to probe the topography
and surface roughness of the films; electrochemical methods
were used to detect reaction intermediates and to determine
the permeability of the films to redox probes; and FTIR and
XPS were used to identify chemical functionalities as a function
of deposition time. Analysis of XPS data by the uniform
overlayer model allowed estimation of dry film thicknesses.
Films deposited for 60 min and 16.5 h were also characterized
as points of comparison for evaluation of the films deposited at
times ≤10 min.
■
MATERIALS AND METHODS
Materials. Dopamine hydrochloride (98%), sodium bicarbonate
(99%), and potassium ferricyanide (99%) were purchased from
Sigma−Aldrich and used as received. Gold films (≈200 nm) used for
XPS analysis were prepared by vapor deposition on Cr-primed (20
nm) Si (100) wafers (Silicon Quest International). The base pressure
in the vacuum chamber for deposition was typically ≈3 × 10−5 Pa (≈5
× 10−7 Torr).
Polydopamine films were prepared by dissolving dopamine
hydrochloride in a 0.1 mol/L carbonate/bicarbonate buffer (pH 8.5)
at a concentration of 1 mg/mL. Substrates were immediately
immersed (vertical orientation) in the solution for 2, 5, 10, or 60
min or 16.5 h deposition times. The samples were then withdrawn,
rinsed thoroughly with water (18 MΩ·cm) to remove unbound
species, and dried with nitrogen.
Atomic Force Microscopy Methods. Polydopamine films were
deposited on template stripped gold (TSG) substrates at deposition
times of 2, 5, 10 and 60 min. TSG substrates were produced according
to the procedure published by Wagner, et al..44 The TSG substrates
are smooth with a measured root-mean-square (rms) roughness of
0.34 nm over a (5 × 5) μm scan area and were also used for reflection
absorption FT-IR spectroscopy. AFM images were obtained using a
XE-100 (Park Systems, Inc.) scanning probe microscope operated in
noncontact mode. The topographic images were recorded at a scan
rate of 1 Hz using a noncontact cantilever (PSIA) with a force
constant of 42 N/m and resonant frequency of ≈320 kHz. Root-mean-
squared roughness values were calculated over the entire scan area
using XEI software (Park Systems, Inc.).
Electrochemical Study Methods. A potentiostat (CHI 660) with
a three-electrode cell was used for all electrochemical studies. The
electrochemical cell had a gold disk working electrode (0.02 cm2), a
platinum wire counter electrode, and a Ag/AgCl reference electrode.
The gold electrode was mechanically polished with a slurry of 1 μm
alumina followed by rinsing with copious amounts of water, and then
electrochemically cleaned in 1 mol/L H2SO4 immediately prior to use.
Polydopamine films used for electrochemical studies were deposited
from 1 mg/mL dopamine in 0.1 mol/L (bi)carbonate buffer (pH 8.5)
on planar gold working electrodes at deposition times of 2, 5, 10, and
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60 min and 16.5 h. All films were removed from the dopamine
deposition solution, rinsed with water, and dried with N2 before being
immersed in the electrochemical cell. A supporting electrolyte of
deoxygenated phosphate-buffered saline (PBS; 0.1 mol/L phosphate
and 0.5 mol/L NaCl, pH 7) was used for all electrochemical
experiments. Potassium ferricyanide (5 mmol/L) was dissolved in the
same phosphate buffer and deoxygenated prior to redox probe
experiments.
Fourier Transform Infrared Spectroscopy Methods. Infrared
reflection absorption spectra were obtained by using an FTIR
spectrometer equipped with a wire grid infrared polarizer (p-
polarized), a variable-angle specular reflectance accessory (reflectance
angle 75°), and cryogenic mercury−cadmium−telluride detector.
Filtered and dried house compressed air was used to purge the
sample compartment of ambient carbon dioxide and water vapor.
Presented spectra (3900−1250 cm−1) are the result of averaging 1024
scans at 4 cm−1 resolution and have been corrected for baseline drift.
All FTIR measurements were performed on freshly prepared samples.
These samples were subsequently used for AFM studies as described
above.
X-ray Photoelectron Spectroscopy Methods. The XPS
measurements were made on a Kratos Axis Ultra DLD spectrometer
with monochromatic Al Kα radiation as the excitation and an X-ray
power of 140 W. All measurements were done in the hybrid mode,
with 90° takeoff angle, step size of 0.1 eV, and dwell time of 298.5 ms.
High-resolution scans were acquired for N 1s, O 1s, C 1s, and Au 4f
(data not shown) regions collected in the fixed analyzer transmission
mode with a pass energy of 40 eV. Binding energies were calibrated
with respect to the Au 4f 7/2 peak at 84.0 eV. Elemental core-level
spectra were fit by use of CasaXPS software. After subtraction of a
linear background for N 1s, O 1s, and C 1s peaks and a Shirley
background for Au 4f peaks, all spectra were fit by use of a convolution
of Gaussian and Lorentzian line shapes with a typical ratio of 70:30.
The peak-fitting procedure used a minimum number of peaks
consistent with the best fit with consideration of peak position, full
width at half-maximum, and intensity.
■
RESULTS AND DISCUSSION
Atomic Force Microscopy Results. AFM was used to
investigate the topography and surface roughness of polydop-
amine films deposited for times of 2, 5, 10, and 60 min.
Template stripped gold (TSG) substrates were removed from
dopamine solutions at various time intervals, rinsed with water,
and blown dry with nitrogen gas before being imaged by AFM.
Representative images of 1.0 × 1.0 μm2 scans are displayed in
Figure 2, and representative images and line scans of 0.5 × 0.5
μm2 scans are displayed in Figures S1−S4 in Supporting
Information.
TSG was chosen as the substrate because of its low surface
roughness.44 The rms roughness (defined as the standard
deviation of the height values over the 1.0 × 1.0 μm scan area)
measured on a freshly prepared TSG sample was 0.3 nm (inset
table of Figure 2, 0 min). The TSG sample surface is
topographically made up of flat islands of gold, bounded by
multiple grain boundaries (Figure 2a). In the analysis of these
images, we are following the hypothesis that the polydopamine
material is formed in solution and adsorbs to the TSG surface.
At short times we would expect less adsorption than at longer
times, and we would also expect to observe larger adsorbed
polymer particles with increasing time of the autoxidation
polymerization reaction. What we observed is that the surface
topography changes notably after immersion in solutions of
dopamine at all time points examined and that the topo-
graphical features match well with the working theory of
polymer growth in solution followed by adsorption. The
topography of the deposited films appears granular (Figure 2b−
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Figure 2. Topographical AFM images (noncontact mode) of polydopamine films deposited on TSG substrates for (a) 0, (b) 2, (c) 5, (d) 10, and (e)
60 min deposition times.
d), as has been observed in other SPM studies of deposited
films of polydopamine34−37 and electrochemically deposited
synthetic melanin.41,42 Line scans reveal that the average
resolvable cross-sectional diameter of the granular features is 25
± 5 nm for the 2 and 5 min samples (Figures S1 and S2,
Supporting Information) and increases slightly to 30 ± 5 nm
for the 10 and 60 min samples (Figures S3 and S4, Supporting
Information). The granular feature size we report are similar to
those observed in an image of a polydopamine film deposited
via multiple dip cycles in fresh basic dopamine solution,
reported by Bernsmann et al..37 The granular features we report
also fall in the range reported for electrochemically deposited
melanin aggregates (≈2−50 nm), but we did not image any fine
features, such as those reported by Orive et al.,42 that would
suggest observation of melanin protomolecules.
In addition to the granular features, there are some larger-
diameter particle features that increase in size and frequency of
occurrence with deposition time. These larger particle features
generally range in size from ≈40 to ≈50 nm in diameter with a
z-dimension that ranged from ≈3 to ≈8 nm. The increase in
frequency of observation of these larger particle features with
deposition time supports the idea that larger polymer particles
that have formed in solution adsorb to the surface at longer
deposition times.
The 2 and 5 min samples have dark features where the AFM
tip has scanned over a lower region of the surface. We attribute
these features to gaps in the deposited polydopamine film. An
approximate thickness of the film, as measured by the depth of
the gaps in the film, was ≈2.5−3 nm for the 2 min sample and
ranged from ≈4 to ≈10 nm for the 5 min sample. These values
are higher than those we estimate by the standard uniform
overlayer model analysis of the XPS data (See X-ray
Photoelectron Spectroscopy Results section and Figure 7)
but are within an order of magnitude and may be higher due to
hydration of the film under ambient conditions.
In order to better estimate the surface coverage for the 2 and
5 min samples, grain analysis image software (XEI version 1.6,
Park Systems, CA) was used to estimate the area of the gaps in
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the film by setting the threshold to exclude features above a
certain height threshold that outlined the perimeter of the gaps
well. This area was subtracted from the scan area and divided
by the scan area in order to estimate the film coverage for
Figure 2b,c. This analysis resulted in ≈80% coverage at 2 min
and ≈95% coverage at 5 min.
The gap features are notably absent in the image of the
sample with a deposition time of 10 min (Figure 2d). Line
scans across lower regions in the 10 min sample revealed a
depth of only ≈1 nm and do not suggest a gap in the deposited
polydopamine film that extends to the TSG surface (Figure S3,
Supporting Information). The topology of the 10 min sample
image suggests a continuous amorphous film with larger
features, which we attribute to larger polymer species adsorbed
from solution. More dramatic changes are observed in the
image of the sample treated for 60 min (Figure 2e). This image
consists roughly of three regions where relatively flat areas of
continuous amorphous film are covered by a second layer of
larger polymer species. The depth of the gaps measured in the
60 min image are ≈5 nm, as seen in the line scans in Figure S4
(Supporting Information). Rms roughness (Rq) values
calculated over the full 1.0 × 1.0 μm2 scan areas shown in
Figure 2 are 1.1, 1.3, 2.1, and 4.4 nm for the 2, 5, 10, and 60
min samples, respectively (inset table in Figure 2).
These AFM images suggest that 10 min is a sufficient
deposition time from a basic stirred solution of dopamine for
achieving a topologically continuous polydopamine film with
maximal film coverage and minimal surface roughness.
Electrochemical Study Results. Cyclic voltammetry was
used to probe the molecular species present in the polydop-
amine film and to assess its porosity. To assist in interpreting
the voltammetric behavior of the polydopamine film, we first
examine the electrochemical response of dopamine (5 mmol/
L) from −0.5 to 0.7 V versus Ag/AgCl at 0.025 V/s in a
deoxygenated supporting electrolyte of PBS (0.1 mol/L
phosphate and 0.5 mol/L NaCl, pH 7) (Figure 3a). On the
initial scan, one anodic peak appears at 0.25 V and two cathodic
peaks at 0.13 and −0.22 V. In subsequent scans there are two
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Figure 3. Cyclic voltammograms of (a) 5 mmol/L dopamine in PBS
(0.1 mol/L phosphate and 0.5 mol/L NaCl, pH 7) and (b) initial
scans of polydopamine films deposited onto gold electrodes for 2, 5,
10, and 60 min and 16.5 h. Representative subsequent scans taken
after the initial cyclic voltammogram are shown for the 10 min sample.
anodic peaks, at −0.17 and 0.33 V, and two cathodic peaks at
slightly more positive voltages than the initial scan. On the basis
of previous electrochemical studies of dopamine, the observed
voltammetric responses are assigned to the dopamine/
dopaminequinone (E1/2 = 0.19 V) and leucodopamine-
chrome/dopaminechrome (E1/2 = −0.20 V) redox couples
(see Figure 1a).18−21 With successive scans, all peak currents
decreased and peak separations increased, observations
consistent with the formation of an electrochemically inactive,
insulating polydopamine film within the potential region
examined.45
Films deposited on gold electrode surfaces from dopamine
solutions at various times were rinsed, dried, and immediately
placed in the electrochemical cell for examination. The
potential was cycled from −0.5 to 0.7 V versus Ag/AgCl at
0.025 V/s in a deoxygenated supporting electrolyte of PBS (0.1
mol/L phosphate and 0.5 mol/L NaCl, pH 7). Trace amounts
of residual dissolved O2(g) and CO2(g) were observed in the 0
min sample at E1/2 = 0.39 and −0.13 V, respectively. Initial
scans recorded on films deposited for 2, 5, 10, and 60 min and
16.5 h are shown in Figure 3b, along with representative
subsequent scans shown for the 10 min sample (a larger plot of
the 10 min time point can be seen in Figure S5, Supporting
Information). Cyclic voltammetry (CV) of deposited polydop-
amine films is now discussed and compared to that of
dopamine. Initial scans on films deposited at short times
(≤10 min) show two anodic peaks (−0.04 and 0.20 V) and one
cathodic peak (0.16 V). The −0.04 V peak current decreases in
successive scans (see 10 min CV example). This peak is
assigned to the oxidation of 5,6-hydroxyindole to 5,6-
indolequinone, which is then incorporated into the polymer
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over the rest of the forward anodic scan.19 The 0.20 V/0.16 V
peak pair (E1/2 = 0.18 V) is assigned to the dopamine/
dopaminequinone redox couple. The presence of the peak at
0.20 V on the initial scan indicates that nonoxidized dopamine
is present in the deposited films that are drawn from solution
and that it is not removed by rinsing. This observation is
consistent with the hypothesis by Bernsmann et al.35 that
nonoxidized dopamine is needed for deposition of dopamine-
melanin (polydopamine) films. Negligible voltammetric activity
at −0.20 V indicates that intermediate species such as
leucodopaminechrome or dopaminechrome are absent or are
at low concentration in the deposited films, or they are present
but are electrochemically isolated due to large insulating
regions. The greatly reduced voltammetric activity of the 16.5 h
sample suggests that the electrode surface has been largely
passivated by an insulating polydopamine film.
The anionic redox couple of ferricyanide/ferrocyanide was
used to examine the porosity of the deposited films as a
function of deposition time. Prior to immersion of polydop-
amine films in Fe(CN)63− solutions, the films were electro-
chemically conditioned by cycling the potential in PBS buffer
between −0.04 and 0.50 V. This step was performed to oxidize
unreacted dopamine in the film, which was found to interfere
with the redox probe signature. Without the conditioning step,
redox probe currents did not vary with deposition time (data
not shown), as was found for the conditioned films as discussed
below. After conditioning, the polydopamine-coated electrode
was removed from the buffer solution, rinsed, dried, and placed
in the same buffer solution containing 5 mmol/L K3Fe(CN)6,
and the potential was scanned between 0.50 and −0.04 V
versus Ag/AgCl (Figure 4). For the 2 min deposition time, the
Fe(CN)63−/Fe(CN)64− redox activity is only slightly dimin-
ished compared to that at a bare gold electrode. This
observation indicates the formation of a nonuniform, porous
polymer film or one that is exceedingly thin. At longer
deposition times (5 and 10 min), peak currents clearly decrease,
Figure 4. Cyclic voltammograms of 5 mmol/L Fe(CN)63− in
deoxygenated PBS supporting electrolyte, recorded at polydopamine
films deposited onto gold electrodes for 2, 5, 10, and 60 min and 16.5
h.
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splitting between the oxidation and reduction peaks increases, Table 1. Infrared Bands of Polydopamine Deposited on
and the overall shape of the CV becomes more sigmoidal. Template Stripped Gold Surfaces
These observations are consistent with the formation of a
wavenumbera (cm−1) assignment
partially blocking polymer layer on the electrode surface in
which the diffusion of Fe(CN)63−/Fe(CN)64− through gaps in Exposure Time 2 min
the polydopamine film is impeded as the gaps decrease in size. 3520 br ν(N−H), ν(O−H)
For films deposited at longer times (60 min and 16.5 h), 2962, 2925, 2852 ν(C−H)
negligible electrochemical activity is observed, suggesting the 1723 w ν(C=O)
formation of a completely blocking polydopamine film on the 1580 br νring(C=C)
gold electrode surface. Similar electrochemical behavior has Exposure Time 5 min
been reported for dopamine-melanin films formed via multiple 3400 br ν(N−H), ν(O−H)
dip cycles in unstirred fresh dopamine solutions34 or by 2962, 2925, 2852 ν(C−H)
immersion in an aerated basic solution of dopamine.21 The 1723 w ν(C=O)
electrochemical data are largely consistent with the AFM results 1580 br νring(C=C)
presented above that show the formation of a continuous Exposure Time 10 min
polymer film after a deposition time of 60 min. Although we 3300 br ν(N−H), ν(O−H)
did not observe any conductive properties of the deposited 2962, 2925, 2852 ν(C−H)
polydopamine film within the potential region examined, it 1723 ν(C=O)
should be noted that semiconductor behavior has been 1580 br νring(C=C)
measured for thick melanin films deposited electrochemically Exposure Time 60 min
from a synthetic melanin solution.42 3300 br ν(N−H), ν(O−H)
Fourier Transform Infrared Study Results. FTIR spectra 2962, 2925, 2852 ν(C−H)
taken in the reflection mode acquired from polydopamine films 1723 sh ν(C=O)
deposited on TSG substrates for 2, 5, 10, and 60 min and 16.5 1596, 1510 νring(C=C), νring(C=N)
h are displayed in Figure 5. These samples are the same as 1460 w sh νring(C=C)
1354 νring(CNC)
Bulk Polydopamine (16.5 h)
3296 br ν(N−H), ν(O−H)
2962, 2925, 2852 w sh ν(C−H)
1720 w sh ν(C=O)
1596, 1510 νring(C=C), νring(C=N)
1460 νring(C=C)
1354 νring(CNC)
a
Abbreviations: br, broad; w, weak; sh, shoulder.

the cyclic voltammetry experiments described above. Spectra

Figure 5. Reflection absorption FTIR spectra of polydopamine films
deposited on TSG substrates for 2, 5, 10, and 60 min and 16.5 h
deposition times. Traces for 2, 5, and 10 min are scaled by 2×, and the
16.5 h trace is scaled by 0.5×.
those analyzed by AFM in Figure 2. Select IR bands are labeled
and listed in Table 1 along with mode assignments, based on
reference infrared band tables46 and prior work.16 Prior IR
studies of melanin polymers, obtained from natural and
synthetic sources, have been reported for bulk polymer
films.13,14,16 Here we report IR analysis of the early stages of
polydopamine film formation. Although spectra are weak for
polydopamine films at short deposition times (≤10 min),
features are observed that indicate the presence of dopamine
and other intermediate species, results that are consistent with
8624
recorded from films deposited for 2 and 5 min are similar and
are discussed together. Features observed are a broad band at
3700−3300 cm−1 that is assigned to ν(N−H) and ν(O−H)
stretching modes; aliphatic ν(C−H) stretching modes at 2962,
2925, and 2852 cm−1; and a broad peak centered at 1600 cm−1
that is assigned to νring(C=C) stretching modes. The positions
of these features are consistent with those observed in reference
spectra of dopamine (data not shown). Because of the low
signal-to-noise ratio of the spectra acquired at 2 and 5 min, we
are unable to unambiguously identify a peak at ≈1700 cm−1
that would indicate the presence of intermediate 5,6-
indolequinone species; however, at longer deposition times
(10 and 60 min spectra), a feature clearly appears at 1723 cm−1
that is assigned to ν(C=O) groups, indicating the presence of
quinone groups. For the 60 min and 16.5 h deposition times,
the 1723 cm−1 feature decreases in relative intensity, indicating
that carbonyl species are a minor component of the bulk
polydopamine film. In the 60 min spectrum, two features at
1596 and 1510 cm−1 emerge from the broad band centered at
1580 cm−1 and are assigned to νring(C=C) and νring(C=N)
stretching modes, respectively, confirming the presence of
aromatic amine species in the deposited film.16 The peak at
1354 cm−1, which appears as a shoulder in the 60 min sample,
is assigned to indole ring CNC stretching modes. The presence
of indole features in the bulk polydopamine film supports the
proposed structure of melanin-like polymers (polydopamine,
dopamine-melanin) consisting of 5,6-dihydroxyindole/5,6-
dx.doi.org/10.1021/la400587j | Langmuir 2013, 29, 8619−8628
Langmuir Article

indolequinone units.17,40,47 The center of the broad peak

observed above 3000 cm−1 shifts from ≈3500 to 3300 cm−1
with increasing deposition time. This shift indicates a loss of
primary amine groups [peaks expected at ≈3500 cm−1 for
ν(N−H)asymmetric and 3400 cm−1 for ν(N−H)symmetric
modes] and the formation of secondary amines (peak expected
at ≈3330 cm−1), as is expected in the formation of
polydopamine.46
X-ray Photoelectron Spectroscopy Results. XPS
provided chemical state information and thickness estimates
of the polydopamine films as a function of deposition time.
XPS has been used previously to estimate the elemental
composition, distribution of functional groups, and thickness of
melanin films.7,35 Clark et al.7 reported high-resolution spectra
of the carbon, nitrogen, and oxygen regions acquired from bulk
samples of eumelanin biopolymers obtained from natural
sources or through autoxidation or enzymatic polymerization of
known precursor starting materials, including dopamine.
Recently, Bernsmann et al.35 reported XPS results from
dopamine-melanin films created via multiple dip cycles in
unstirred fresh dopamine solutions. We report here XPS
analysis of the early stages of polydopamine film formation at
deposition times ≤10 min.
XPS measurements were made on polydopamine films
deposited for times of 2, 5, 10, and 60 min and 16.5 h. XPS
signatures originating from C, O, and N atoms within the films
were measurable on all samples. Representative high-resolution
spectra of the C 1s, O 1s, and N 1s regions, obtained on a
polydopamine film deposited for 10 min, are shown in Figure 6.
Region scans for all elements detected and at all deposition
times are presented as Supporting Information. The energies
and assigned chemical groups for each peak within the region
envelopes are listed in Table 2 and are similar to those reported
by Clark et al.7 The percent contribution for each functional
group within the C 1s, O 1s, and N 1s regions are presented in
Table 3. Generally, the peak energies of each component
remained constant but the relative intensities of each Figure 6. High-resolution XPS spectra of C 1s, N 1s, and O 1s regions
component varied, particularly at times <10 min. The C 1s for a polydopamine film deposited from solution for 10 min.
region is fit with four peaks assigned to CHx/C−NH2, C−O/
C−N, C=O/C=N, and π → π* species. The basic peak shapes Table 2. XPS Peak Binding Energy Assignmentsa
remain constant, with the dominant peaks being the attributed functional group binding energy (eV)
to CHx/C−NH2 and C−O/C−N species. At short times the CHx, C−NH2 284.4 ± 0.1
fraction of CHx/C−NH2 species is higher than that of C−O/ C−O, C−N 285.6 ± 0.2
C−N species; at longer times, the ratios are reversed. This C=O 287.7 ± 0.4
observation can be explained by the conversion of the primary π → π* 290.9 ± 0.3
amine-containing species, dopamine, into reaction intermedi- O=C 531.0 ± 0.2
ates and tautomers (secondary and tertiary amine species) O−C 532.9 ± 0.1
shown in Figure 1. The contribution from carbons doubly =N−R 398.6 ± 0.1
bonded to oxygen varies from 14% to 18% and is attributed to R−NH−R 399.9 ± 0.1
the intermediate species and their possible tautomers (Figure R−NH2 401.9 ± 0.3
1b). The π → π* shakeup satellite of the C 1s peak is a a
Uncertainties were calculated as 1 standard deviation of at least three
common energy loss feature for aromatic carbon species and replicates.
first appears in the 10 min deposition sample. This feature
increases in relative intensity with deposition time (Figure S6,
Supporting Information).48 feature, originating from sodium incorporated into the film at
The O 1s region is fit with two peaks assigned to O=C and this long deposition time (Figure S8, Supporting Information).
O−C species. The fraction of each species remains constant The N 1s region is fit with three peaks assigned to primary
with deposition time, with the O 1s signal composed of ≈30% (R−NH2), secondary (R−NH−R), and tertiary/aromatic
O=C and ≈70% O−C. The O=C and O−C components are (=N−R) amine functionalities.7,35 On the basis of the chemical
found in dopamine (O−C only), the intermediate/tautomer structures of dopamine, accepted intermediate species, and the
species, and the polydopamine material. The O 1s spectrum of most recently proposed structures of polydopamine,8,40 the
the polydopamine film deposited for 16.5 h exhibited an primary amine is associated with dopamine, the secondary
additional peak at 535.4 eV that is attributed to a Na KLL amine is associated with both intermediate species and
8625 dx.doi.org/10.1021/la400587j | Langmuir 2013, 29, 8619−8628
Langmuir
Table 3. XPS Functional Group Percentages as a Function of Deposition Time (±6% RSD)
C 1s
deposition time CHx, C−NH2 C−O, C−N C=O π → π*
2 min 48 31 18 3 35
5 min 45 36 15 3 33
10 min 43 39 14 4 29
60 min 39 42 14 5 28
16.5 h 34 44 18 4 32
polydopamine, and the tertiary amine is associated with
tautomeric species of the intermediate species 5,6-dihydrox-
yindole and 5,6-indolequinone. The relative percentages of
these amine species vary with deposition times. At the shortest
deposition time, 2 min, the relative amount of tertiary/aromatic
amine groups associated with polymerization reaction inter-
mediates is the greatest, followed by the secondary amine signal
and then the primary amine. With increasing deposition time
and film thickness, the secondary amine component grows and
dominates the N 1s region, as it is associated with
polydopamine. The contribution from the primary amine
group is minor at 2 and 5 min deposition times but contributes
between 15% and 18% at times ≥10 min to the total N 1s
signal.
It is interesting to note that at deposition times ≥10 min, the
relative fractions of each of the amine species remain constant.
This suggests that the chemical environment of the surface of
the polydopamine film is comparable at times ≥10 min and that
intermediate species and dopamine molecules are present at the
surface of the polymer film.
The thickness of the deposited polydopamine film was
estimated according to the standard uniform overlayer model:
IAu = IAu0 exp (−t/LAu), where IAu0 is the measured intensity of
the Au 4f 7/2 peak measured on a clean sample of gold, IAu is the
measured intensity of the Au 4f 7/2 peak in gold samples
exposed to the dopamine deposition solution, t is the thickness
of the adsorbed polydopamine film, and LAu is the average
practical effective attenuation length of Au 4f 7/2 photoelectrons
in the polydopamine overlayer. The value of LAu was calculated
by use of the NIST Standard Reference Database 82: NIST
Electron Effective-Attenuation-Length Database (version 1.1)
to be 3.45 nm, using the following values: molecular formula of
polydopamine monomer unit = C8H4NO2; number of valence
electrons (Nv) = 53; density = 1.3 g/cm3;35 and band gap ≈1
eV.49 The thickness of the adsorbed polydopamine film
determined by attenuation of gold intensity measured by XPS
as a function of deposition time is plotted in Figure 7. At short
deposition times (≤10 min), the film thickness increases
approximately linearly. At deposition times ≥10 min, the
deposition rate decreases, which can be explained by the
consumption of reactant (dopamine) in the solution as the
reaction proceeds. This observation was also reported by
McCloskey et al.32 and reflects the consumption of dopamine
by polymerization in solution versus deposition at the surface of
interest. The thickness we measure at the 1 h time point, ∼8−
10 nm range, compares well with that reported by Bernsmann
et al.36 for a dopamine film deposited from an aerated solution.
■
CONCLUSIONS
In this study, we focused on the examination of polydopamine
films deposited at times ≤10 min from stirred solutions of
dopamine, with the goal of determining the minimum
deposition time necessary to achieve a continuous polydop-
8626
Article
O 1s N 1s
O=C O−C =N−R R−NH−R R−NH2
65 49 41 10
67 31 61 8
71 10 72 18
72 10 76 14
68 11 74 15
Figure 7. Thickness of polydopamine films determined from
attenuation of Au 4f XPS signal intensity as a function of deposition
time. Error bars represent 1 standard deviation of at least three
replicates.
amine film with physicochemical characteristics similar to films
deposited under the same conditions but for longer times. The
XPS, FTIR, AFM, and electrochemical data acquired from films
deposited at all times examined are consistent with previously
published analyses of melanin polymers derived from
dopamine. Chemical signatures of starting material species
(dopamine), intermediate species (tertiary/aromatic amine
containing tautomers of 5,6-hydroxyindole and 5,6-indolequi-
none), and polydopamine were detected in various relative
amounts. At the 2 and 5 min deposition times, chemical
signatures from dopamine and intermediate species dominated
the data; and at times ≥10 min, chemical signatures from
polydopamine dominate the data. The chemical species
identified in this study confirm the contemporary reaction
mechanism of polydopamine formation by autoxidation of
dopamine and also suggest that tautomers of 5,6-hydroxyindole
and 5,6-indolequinone play a major role in the reaction.
Regarding the structure of polydopamine, the data presented
here cannot definitively confirm proposed structures,8,40 but
our data do confirm the presence of starting material
(dopamine) and intermediate species (5,6-hydroxyindole and
5,6-indolequinone) at the surface of polydopamine films
deposited from solution.
On the basis of the data presented here, we conclude that a
minimum deposition time of 10 min is required to form a
continuous film of polydopamine from a stirred solution with
the substrate held in a vertical orientation. The topography of a
film formed under these conditions appears largely continuous
with a minimum amount of larger polymeric particles. It should
be noted that pinhole-free films, as measured with an anionic
dx.doi.org/10.1021/la400587j | Langmuir 2013, 29, 8619−8628
Langmuir
ferricyanide redox probe, require deposition times ≥60 min.
Identification of these conditions is important in advancing the
use of polydopamine as a modification layer for various surface,
optical, and nanomaterial measurement methods.
■
ASSOCIATED CONTENT
*
S Supporting Information
Nine figures showing 500 nm × 500 nm AFM images and line
scans measured on polydopamine films deposited for 2, 5, 10,
and 60 min; enlarged graph of Figure 3b, 10 min time point;
and high-resolution XPS spectra of C 1s, N 1s, O 1s, and Au 4f
regions collected on polydopamine films deposited for
increasing times on gold substrates. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail rebecca.zangmeister@nist.gov; telephone 301-975-
4912; fax 301-975-2643.
Present Address
† doped polyindole. J. Phys. Chem. Solids 1996, 57 (6−8), 1145−1151.
T.A.M.: Department of Chemistry & Biochemistry, University
of Mississippi, University, MS 38677.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
T.A.M. acknowledges the NRC/NIST postdoctoral fellowship
program for support while this research was conducted. Certain
commercial equipment, instruments, or materials are identified
in this document. Such identification is not intended to imply
recommendation or endorsement by the National Institute of
Standards and Technology, nor is it intended to imply that the
products identified are necessarily the best available for the
purpose.
■
ABBREVIATIONS
DA, dopamine; L-DOPA, 3-(3,4-dihydroxyphenyl)-1-alanine;
DHI, 5,6-dihydroxyindole; XPS, X-ray photoelectron spectros-
copy; AFM, atomic force microscopy; FTIR, Fourier transform
infrared spectroscopy
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