Analytica Chimica Acta 554 (2005) 43–51

Impedance spectral studies of self-assembly of alkanethiols with different

chain lengths using different immobilization strategies on Au electrodes
Shinn-Jyh Ding a , Bin-Wha Chang b , Ching-Chou Wu c ,
Min-Feng Lai d , Hsien-Chang Chang d,e,∗
a Institute of Oral Materials Science, Chung-Shan Medical University, Taichung 402, Taiwan
b Department of Healthcare Administration, Hung-Kuang University, Taichung 433, Taiwan
c Department of Bio-Industrial Mechatronics Engineering, National Chung-Hsing University, Taichung 402, Taiwan
d Institute of Biomedical Engineering, National Cheng-Kung University, No. 1, Ta-Hseuh Road, Tainan 701, Taiwan, ROC
e Center for Micro/Nano Technology Research, National Cheng-Kung University, Tainan 701, Taiwan

Received 28 May 2005; received in revised form 17 August 2005; accepted 17 August 2005
Available online 19 September 2005

Abstract
Alkanethiols are commonly used self-assembly reagents in the preparation of modified electrode. In this study, self-assembled monolayers (SAMs)
of alkanethiols with different chain lengths formed on gold electrode were mainly investigated by electrochemical impedance spectroscopy (EIS) in
addition to cyclic voltammetry (CV). Two types of immobilization strategies applied to the electrode were also examined. The interfacial properties
of all self-assembled electrodes were evaluated in the presence of Fe(CN)6 3−/4− redox probe. A simple equivalent circuit model with a constant
phase element was used to interpret the obtained impedance spectra. The results of cyclic voltammetry revealed that the voltammetric behavior
of the redox probe was influenced by the electrode surface modification. The EIS data indicated that the formation of saturated SAMs on gold
required at least 30 min and more apparent differences occurred with immobilization time compared to that obtained by CV. Depending on the
alkyl chain length of the SAMs, the electron-transfer resistance increased and the capacitance decreased. It was found a good linear relationship
between inverse capacitance and chain length for carboxyl-terminated SAMs. To achieve a defect-free structure and/or close packing, the direct
application method may provide a more effective, rapid, and facile self-assembly immobilization than immersion method from the viewpoint of
the electron-transfer resistance.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Self-assembly; Electrochemical impedance spectroscopy; Equivalent circuit; Cyclic voltammetry; Alkanethiol

1. Introduction functionalized self-assembled organic monolayers of alkanethi-

Self-assembled monolayers (SAMs) provide a means for the
molecular design on a bioinert surface with specific functions.
Therefore, immobilization techniques for SAMs, particularly
those of alkanethiolate on gold electrodes, have become the
focus of intensive investigations [1–6]. SAMs technology has
been widely applied in the immobilization of molecular recog-
nition layer on electrochemical, surface plasmon resonance-
based and (electrochemical) quartz crystal microbalance-based
biosensors [1,4,7–9], as well as in surface modification of bio-
materials [10,11]. McClary et al. reported the use of terminally
∗ Corresponding author. Tel.: +886 6 2757575x63426; fax: +886 6 2343270.
E-mail address: hcchang@mail.ncku.edu.tw (H.-C. Chang).
ols on gold to modulate extracellular matrix-dependent cell
attachment and spreading [11]. SAMs of co-mercaptocarboxylic
acid termini on gold electrodes were utilized by Malem and Man-
dler as a sensing tool to detect the dopamine neurotransmitter
in the presence of ascorbic acid [1]. Pang et al. presented an
electrochemical method for determining the amount of DNA
covalently attached to a monolayer of thioctic acid immobilized
on an electrode surface [9]. These previous studies suggested that
a self-assembly of ␻-mercaptocarboxylic acid monolayer may
provide a very promising basis for immobilizing biomolecules
via its functional group. More importantly, it is possible to
apply different functionalized SAMs to cross-link with targeted
molecules such that there is very little disruption to self-assemble
process or destabilization of the SAMs selected in specific appli-
cations [2,5,6,10]. Keselowsky et al. found that SAMs with

0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2005.08.046
44 S.-J. Ding et al. / Analytica Chimica Acta 554 (2005) 43–51
terminal CH3 , OH, COOH, and NH2 functionalities altered the
functional presentation of the major integrin binding domain of
adsorbed fibronectin and modulated integrin binding, localiza-
tion, and specificity [10].
A variety of techniques, including X-ray photoelectron,
infrared, and Raman spectroscopies, contact angles, electro-
chemistry, and atomic force microscopy were used to study
characteristics of alkanethiol-modified electrode [8,12–14].
Electrochemical impedance spectroscopy (EIS) is a sensitive
and non-destructive technique and widely used for characteri-
zation of biomaterial-functionalized electrodes and biocatalytic
transformations at electrode surfaces, and specifically for trans-
duction of biosensing events at electrodes [15–17]. For example,
Pei et al. amplified the response signal of antigen–antibody inter-
actions based on biotin labeled protein–avidin complex on a
SAMs-modified electrode, which was evaluated by impedance
spectroscopy [16]. In a recent study, we combined the electro-
chemical impedance spectroscopy and self-assembled layer as
a means of quantitating the avidin–biotin interaction [18].
To study the integrity of SAMs, we presented an electro-
chemical detection of carboxyl-terminated alkanethiol mono-
layers that have different chain lengths in a solution
of 2-(N-morpholino) ethanesulfonic acid (MES) containing
Fe(CN)6 3−/4− redox couple by means of EIS and cyclic voltam-
metry (CV). By applying two different immobilization proce-
dures including immersion and direct application methods, the
formation process of self-assembly was also evaluated in detail
by EIS. Subsequently, data were modeled and interpreted by
a simplified equivalent circuit with a constant phase element,
and then the model parameters were estimated from complex
non-linear least-squares (CNLS) method fitting to experimental
data.
2. Experimental
2.1. Preparation of thiol SAM
Carboxyl-terminated alkanethiols including 4,4-
dithiodibutyric acid ([–S(CH2 )3 COOH]2 , 4-DTBA), 11-merca-
ptoundecanoic acid (HS(CH2 )10 COOH, 11-MUA), and 16-
mercaptohexadecanoic acid (HS(CH2 )15 COOH, 16-MHA), all
purchased from Aldrich, were used without further purification.
Deposition solutions of 5 mM final concentration were prepared
by dissolving the corresponding reagent in absolute ethanol.
The electrolyte was 50 mM MES solution (pH 6.0) buffered
with 1 M NaOH. All solutions were prepared with pure water
purified through a Milli-Q system (Mllipore, Bedford, MA).
All other chemicals were of reagent grade and used as received.
Before experiments, all solutions and chemicals were stored
at 4 ◦ C. The Au electrode of 1.6 mm diameter was polished
with 0.03 ␮m alumina slurry, and then ultrasonically cleaned
in distilled water and in absolute ethanol. After drying in
air, the clean Au electrode was immediately immersed in the
deposition solution for different predetermined time periods at
room temperature, followed by rinsing off the residual thiol
molecules with ethanol and drying in air. In contrast to the
above-used immersion method, a direct application method, as
reported by Akram et al. [19], was used for comparative study.
In the direct application method, 20 ␮L of 5 mM deposition
solution was placed onto the clean electrode for 20 min. After
that, the electrode was ultrasonically treated in ethanolic
solution for 20 s to remove any unbound thiols and then air
dried.
2.2. Surface coverage measurement
In addition to impedance measurements, the surface coverage
of SAM-modified electrodes was used to evaluate the differences
in characteristics of molecular layers assembled on a Au elec-
trode by immersion and direct application methods. The surface
coverage was determined using the expression Q = nFAΓ , where
Q is the amount of charge (C), Γ the monolayer surface cov-
erage (mol cm−2 ), n the number of electrons involved in the
electron-transfer process (alkanethiol), A the electroactive sur-
face area (cm2 ), and F is the Faraday constant. By measuring the
peak current in a cyclic voltammogram, the electroactive surface
area of a bare gold electrode was determined according to the
Randies–Sevcik equation [20]:
Ipa = (2.69 × 105 )n3/2 AD1/2 ν1/2 COX
where Ipa is anodic peak current in A cm−2 , COX the bulk con-
centration of an oxidant in mol cm−3 , D the diffusion coefficient
in cm2 s−1 , n the number of electrons transferred, and ν is the
potential scan rate in mV s−1 . Among these parameters, a D
value of 0.65 × 10−5 cm2 s−1 at 20 ◦ C [21] was used for a sys-
tem containing 5 mM Fe(CN)6 4− in 0.1 M KCl as supporting
electrolyte.
It is known that when a SAM-modified electrode is placed in
an alkaline solution (pH ≥ 11), the monolayer film will desorb
from the electrode surface via a one-electron reductive path. This
can be represented by the equation:
AuS(CH2 )n X + e− → Au(0) + − S(CH2 )n X,
where X = CH3 , COOH, OH
Hence, the amount of charge arising from SAMs was determined


by integrating the reduction current against time (Q = i dt).
Using the above expression, the surface coverage per unit area
on the SAM-coated electrodes can be easily obtained.
2.3. Electrochemical measurements
All electrochemical experiments were performed using an
IM-6 impedance analyzer (Zahner, Elektrik, Kronach, Ger-
many). Unless mentioned, the 50 mM MES solution containing
2.5 mM Fe(CN)6 3−/4− (1:1) redox couple was used as the sup-
porting electrolyte in electrochemical measurements. CV was
carried out in a conventional three-electrode cell. Ag/AgCl
and platinum wire were used as a reference electrode and a
counter electrode, respectively. Potential was scanned from −0.2
to +0.6 V at a rate of 100 mV s−1 . Impedance measurements
were performed in a two-electrode electrochemical system. A
5 mV amplitude sinusoidal wave was applied at the half-peak
potential (0 V) of the redox couple in the tested frequency
 S.-J. Ding et al. / Analytica Chimica Acta 554 (2005) 43–51 45

range of 1 Hz–100 kHz. All measurements were performed at
25 ± 0.1 ◦ C by a thermostatically controlled water bath and in
triplicate. The IM-6ATHALES software package was used for
acquisition and analysis of impedance spectra, and simulation
of equivalent circuit. One-way ANOVA statistical analysis was
used to evaluate the statistical significance of the fitted circuit
elements. In all cases, results were considered statistically dif-
ferent when p < 0.05.
3. Results and discussion
3.1. Electrochemical characterization of SAMs
The changes on carboxyl-terminated SAM-modified elec-
trodes with immobilization time were investigated in situ using
CV and EIS. The redox couple Fe(CN)6 3−/4− was preferred to
be used as an electrochemical probe for examining the integrity
of an SAM on gold, such as the pinhole degree or defect struc-
ture, insulating properties and the density of the adsorbed layer
[3,14,16,18]. In Fig. 1, well-defined characteristic waves of the
couple can be observed on the bare Au electrode. It was obvious
that the anodic and cathodic current at a gold electrode decreased
with longer adsorbing time of SAMs on the electrode surface
because of the blocking effect of SAMs. In addition, the peak
current decreased more rapidly following a shorter immersion
duration (=1 min) compared to that obtained at a bare electrode.
In contrast, the decrease in peak current was less pronounced
following a longer immersion duration (=30 min) compared to
those obtained in shorter immersion duration. The observed dif-
ferences were more significant when SAMs with a longer carbon
chain was used, implying a rapid formation process of these
SAMs. Regarding the response of 4-DTBA-deposited gold elec-
trode, the peak current decreased in 1 min immobilization and no
further distinct changes appeared with increasing immobiliza-
tion time. This is possibly because the shorter chain monolayer
was a defect structure with lower packing density and coverage
[1,3]. As for the case of 11-MUA, there was a dramatic difference
between the current–potential responses for Fe(CN)6 3−/4− in
the initial 1 min monolayer-covered and the bare gold electrode.
After an immobilization time of more than 10 min, there was no

Fig. 1. Cyclic voltammograins of: 4-DTBA (a), 11-MUA (b), and 16-MHA (c) self-assemblies after different immobilization periods of time on the Au electrodes.
The supporting electrolyte is 50 mM MES containing Fe(CN)6 3−/4− couple. The scan rate is 100 mV s−1 in the potential range from −0.2 to +0.6 V.
46 S.-J. Ding et al. / Analytica Chimica Acta 554 (2005) 43–51

apparent reaction of Fe(CN)6 3−/4− observed at 11-MUA-coated
electrodes, possibly due to the increased thickness and/or dense
structure of the modification layers. The long-chain molecules of
11-MUA not only acted as the electron-transfer barrier but also
as a capacitor. Hence, the cyclic voltammetric curve showed
a large “double layer” region from −0 to +0.4 V [3,22]. Also,
16-MHA exhibited a similar behavior. For long-chain SAMs
molecules, such as 11-MUA or 16-MHA molecules, the tun-
neling of electrons was adversely hindered, resulting in the CV
curves of capacitive shapes instead of electrochemical reversible
shape [14]. While in the case of short-chain SAMs-modified
electrode, the tunneling of electron was less hindered. This
phenomenon was further confirmed by the results of EIS mea-
surements.
The immobilization of substances, e.g. enzymes, antigens/
antibodies or DNA on electrodes always alters the interfa-
cial capacitance and electron-transfer resistance of the con-
ductive electrodes [2,15,16]. The complex impedance plots of
the three COOH-terminated SAMs in MES solution containing
Fe(CN)6 3−/4− for different immobilization times are shown in
Fig. 2. The impedance plot of the bare Au electrode exhibited a
much smaller semicircular diameter ascribed to electron-transfer
resistance than those observed on all SAM-modified electrodes.
The smaller semicircular diameter indicated the faster electron-
transfer kinetics of Fe(CN)6 3−/4− probe on Au electrode. It
can be seen that the semicircular diameter enlarged with the
increase in immobilization time for all COOH-terminated self-
assemblies, implying a high electron-transfer resistance, possi-
bly due to the increased thickness and compactness as well as
the decreased pinhole defects of modifier to the electrode sur-
face [23]. In particular, the two long-chain SAMs of 11-MUA
and 16-MHA had similar EIS behavior in that a large and slight
increasing semicircular diameter after 10 min immobilization,
and presented the greater impedance changes than 4-DTBA. The
semicircular diameter became gradually large with the increas-
ing immobilization time for short-chain 4-DTBA. The result
suggested that the electron-transfer rate of redox probes was
gradually retarded possibly due to the increase of coverage-ratio
of 4-DTBA SAMs. Therefore, the increasing tendency of semi-
circular diameter may explain the decrease in peak current in
CV with longer immobilization duration.
The difference in impedance before and after the SAMs were
immobilized on a Au electrode surface manifested the effective
blocking ability of SAMs to the redox reaction of Fe(CN)6 3−/4− .
Besides, the self-assembled layers with COOH terminal groups
on the electrode surface generated a negatively charged surface

Fig. 2. Nyquist plots (Zim vs. Zre ) of: 4-DTBA (a), 11-MUA (b), and 16-MHA (c) self-assemblies after different immobilization periods of time on the Au electrodes
at the zero potential with the frequency range from 1 Hz to 100 kHz. The supporting electrolyte is 50 mM MES containing Fe(CN)6 3−/4− couple.
 S.-J. Ding et al. / Analytica Chimica Acta 554 (2005) 43–51 47

that reduced the ability of the probing electrolyte to penetrate

the layer, eventually eliminated effectively the response of the
Fe(CN)6 3−/4− anion [16]. Therefore, Warburg impedance can
be neglected when the electrode surface has a complete self-
assembled layer. It was also found that with the formation of
SAMs over a gold surface, the electrochemical reaction hin-
drance increased rapidly in the initial stage, and tended to be
stable in 60 min. As compared to CV, the results of EIS presented
more appreciable differences among SAMs-covered electrodes
for different immobilization time, indicating EIS more sensitive.

3.2. Equivalent circuit model

In order to obtain the values of the electron-transfer resistance

(Ret ) and the interfacial capacitance, the impedance spectra were
fitted by a simple Randle circuit made up of a resistor, electrolyte
resistance (Rs ), in series with an RC parallel circuit (Fig. 3a). A
constant phase element (CPE) is used instead of a pure capac-
itor to compensate for the non-ideal capacitive response of the
interface [18,24]. Data fitting was done using THALES software
with a complex non-linear least-squares method. The quality of
fitting to the equivalent circuit was evaluated by the error value.
In this study, the capacitance (CPE) contained contributions of
both the modified layer capacitance (Cm ) and the double layer
capacitance (Cdl ) [25]. The two capacitances were in series to
each other; hence, the equivalent capacitance was given by the
following expression:
1 1 1 tance of self-assembled monolayers with COOH termini after
= =
CPE Cm Cdl
Similarly,
Ret = Rm + Rdl
where Rm and Rdl are the modified layer resistance and double
layer resistance, respectively. Based on the model, good agree-
ments, in terms of the magnitude and phase angle of impedance
over the 1 Hz and 100 kHz frequency range were achieved
between the simulated and experimental results obtained at an
11-MUA-coated electrode immersed in MES + Fe(CN)6 3−/4−
solution for 30 min (Fig. 3b). Similar results were reported in
a previous study [18] involving EIS evaluation of avidin–biotin
interaction on SAMs-coated electrodes. The obtained error value
is as small as 0.3% that originated from the difference between
experimental result and simulated data, thereby indicating the
good fitting. Hence, the equivalent circuit model with a CPE was
used to simulate the experimental impedance spectra throughout
Fig. 3. Equivalent electrical circuit diagram (a) of the electrochemical interface
used to fit the impedance spectra (b) for the 11-MUA-modified electrode: Ret ,
electron-transfer resistance; Rs , electrolyte resistance; CPE, constant phase ele-
ment instead of Cdl . Asterisk lines present computer fitting of the impedance
spectra using the equivalent circuit as shown above.
the study. The fitted circuit elements Ret , Rs , and CPE capaci-
different immobilization time are listed in Table 1. The results
indicated only a small variation in the Rs values. The capacitance
and Ret of SAMs showed a dependence on the alkanethiol used in
the deposition solution. Essentially, the more time the molecules
are adsorbed on electrode surface, the larger the electron-transfer
resistance.
Ret represents the interfacial electron-transfer rate of the
redox probe between the electrolyte and electrode. In the Nyquist
plot, the diameter of the semicircle at high frequencies corre-
sponds to the magnitude of Ret in an equivalent circuit. Its values
varied when different substances were deposited on the electrode
surface. The Ret value (ca. 1 k) of the bare gold electrode was
very small compared with the values of Ret after the following
steps. With the self-assembly of 4-DTBA to the surface of the
electrode, Ret was expected to increase due to the inhibition of
the electron-transfer by the self-assembled layer. Indeed, there
was a much higher Ret value of 6.1 k at initial 1 min immobi-
lization. After 120 min immobilization the Ret value increased

Table 1
Fitted circuit elements of self-assembled monolayers after different immobilization time on Au electrodes
Time (min) 4-DTBA 11-MUA 16-MHA

Ret (k) CPE (nF) Rs () Ret (k) CPE (nF) Rs () Ret (k) CPE (nF) Rs ()

1 6.1 490.7 875.3 56.1 388.5 877 44.8 294.1 895.8

10 8.6 483.6 889.5 156.8 299.4 846.3 183.1 145.8 827
30 8.4 478.3 875.1 320.3 254.5 887.2 384.8 150.9 868.7
60 10.4 489.7 870.2 396.8 224.1 883.4 561.5 162.3 912.8
120 11.8 489.4 881.9 449.8 198.3 874.3 585.4 161.0 916.7
48 S.-J. Ding et al. / Analytica Chimica Acta 554 (2005) 43–51

to 11.8 k. The Ret values of all samples increased with immo-
bilization time reaching steady-state values at about 60 min. The
Ret value recorded followed the trend: 4-DTBA < 11-MUA < 16-
MHA that positively correlated to chain length. The Ret com-
ponent of the equivalence circuit can be attributed to pinholes
in the monolayer structure. The Ret of 4-DTBA-modified elec-
trode only exhibited a slight increase, indicating that the number
and size of the pinholes did not apparently decrease even after
immersing for a longer time. Additionally, compared with the
other two long-chain SAMs, the 4-DTBA-modified electrode
had much smaller Ret values. The phenomenon may be attributed
that the chain length of 4-DTBA did not effectively block the
electron tunneling. However, the Ret tended to increase on the
sensing surface of long-chain 11-MUA and 16-MHA. This can
be because the packing of the monolayers becomes more com-
pact.
In contrast, the changes in the capacitance determined
from the experimental impedance spectra did not directly
depend on immobilization time. On modified gold surfaces
with 11-MUA and 16-MHA after the initial 1 min modification,
the electrode–electrolyte capacitance gave rise to a dramatic
decrease and tended to be stable in 10 min immobilization. The
capacitance showed no sign of decrease around 490 nF for 4-
DTBA. It is apparent that the capacitance values obtained from
the short-chain 4-DTBA molecules are significantly (p < 0.05)
higher than those observed from the other two SAMs with long
alkyl chain. Due to the shorter chain of 4-DTBA, it formed a
thinner layer on the electrode surface that related to capacitance
compared to 11-MUA and 16-MHA [6].

3.3. Relationship between fitted impedance values and

chain length

To further clarify the significance of the fitted impedance

values, the calibration plots of the Ret and inverse capacitance
were plotted against the chain length of the self-assembly after
a 60 min immobilization time. Fig. 4a shows the derived cali-
bration plot of the Ret versus the chain length with a squared
correlation coefficient of R2 = 0.94 (N = 3). This result clearly
demonstrated that the Ret was not linearly proportional to the
chain length. On the contrary, a better linear relationship between
reciprocal capacitance (CPE−1 ) value and the number of carbon
atoms (n) was found over the present range because of R2 = 0.99
(Fig. 4b). The line is a least-squares fit to the data and the dashed
portion of the line represents an extrapolation to n = 0. The non-
zero intercept of CPE−1 was due to the capacitance provided by
the thiol head group and was similar to that observed by Jen-
nings et al. [26] for SAMs on cupper. The reciprocal capacitance
value was directly related to the number of carbon chain length
that substantiates the major features of the EIS data. Though not
as extensively examined, we also suggested that the reciprocal
capacitance increased linearly with increasing chain length of
the adsorbate, in agreement with the previous studied [3,26].
These results suggested that the alkyl chain length was likely to
govern the kinetics of electron-transfer. According to the liter-
ature [1], longer ␻-mercaptocarboxylic acid monolayers were
required to form better and more highly charged monolayers,
Fig. 4. The relationship of the electron-transfer resistance (Ret ) (a) and reciprocal
capacitance (CPE−1 ) (b) on the 60 min SAM-modified Au electrodes and the
length of the alkyl chain. The number in parentheses is the error value.
whereas shorter monolayers were preferred in order to increase
the rate of electron-transfer. The self-assembled monolayers
allowed a great deal of flexibility with respect to several appli-
cations depending on their terminal functionality (hydrophilic
or hydrophobic control) or by varying the chain length (distance
control) [6]. Many studies indicated the effect of the carbon chain
length on the packing density, intermolecular environment, and
geometry of the self-assemblies [2,3,26]. The longer alkyl thiols
may pack more densely than the shorter ones. Greater packing
densities would lead to higher resistance and greater measured
thickness than expected from the short-chain thiols.
3.4. Comparison of modification methods
The effect of different immobilization strategies including
immersion and direct application methods on integrity of the
 S.-J. Ding et al. / Analytica Chimica Acta 554 (2005) 43–51 49

Table 2
Fitted circuit elements of the three COOH-terminated self-assembled monolayer immobilized with two different methods on Au electrodes
Ret (k) CPE (nF)

Immersion Direct application Immersion Direct application

4-DTBA 8.3 ± 1.3 18.1 ± 0.2 532.6 ± 45.8 439.5 ± 6.5

11-MUA 465.3 ± 16.8 1221.7 ± 111.4 212.6 ± 19.8 222.7 ± 38.9
16-MHA 573.5 ± 16.9 1716.0 ± 121.6 161.7 ± 9.1 161.2 ± 5.7

SAMs was also examined. Based on the results mentioned above solution (pH 13) taken with a bare gold electrode and with gold
and the earlier study [18], EIS is more sensitive than CV in evalu- electrodes modified with 11-MUA using immersion method and
ation of SAMs. Thus, the two different immobilization strategies direct application method. The scan rate is 100 mV s−1 in the
were differentiated using the changes in the fitted impedance potential ranging from −1.4 to −0.2 V. The amount of the cor-
value. In the immersion method, the electrode was immersed responding charge required for the reduction of this monolayer
in thiols solution for 60 min at which time SAMs could tend can be measured by integration of the reduction peak between
to a steady state according to the previous results. As for the −1.0 and −1.2 V in a cathodic scan with subtraction from back-
direct application method, it took 20 min for achieving the self- ground current of the bare Au electrode.
assembly onto the bare electrode. Concerning immobilization Prior to calculate the electroactive surface area of a bare
method effect, detailed analysis of the data reveals that the major Au electrode, the influence of the scan rate on the Au elec-
differences are the electron-transfer resistance values, present- trode was taken into account. In Fig. 6, the changes in cyclic
ing statistically significant differences (p < 0.05) between the voltammograms of the bared Au electrode in the solution of
immersion method and the direct application method, as shown 0.1 M KCl + 5 mM Fe(CN)6 4− were presented with different
in Table 2. The use of the Ret was attractive from an application scan rates from 20 up to 300 mV s−1 in a potential range of
perspective because different modification methods can yield −0.2 to +0.6 V. Although the anodic and cathodic waves are not
different Ret , not similar CPE values. It suggested that a gold full overlappings of each other because larger scan rates lead
surface covered with a long-chain alkanethiol layer with direct to larger currents, a good reversibility in the redox process of
application method blocked almost all Faradaic currents and was the gold electrode can be still observed. Similar behaviors were
highly insulating with an equivalent transfer resistance of 1221.7 observed for the other systems studied [4,20,29]. For example,
and 1716.0 k, respectively, for a surface covered with 11-MUA Kulys et al. found a reversible diffusion-controlled process of
and 16-MHA, much higher than those obtained by immersion phenoxazine derivatives at a bare gold electrode in 0.01 M phos-
method. As expected, Ret was higher for 16-MHA than for the phate buffer containing 0.1 M NaCl between 200 and 600 mV
omer two SAMs. Similarly, a layer of 4-DTBA was much less with different scan rate. The anodic peak current of phenoxazine
insulating with Ret of 18.1 k but greater compared to 8.3 k
of corresponding immersion-assembled monolayers.
The electron-transfer behavior of the SAMs is essentially
related to the surface morphology and defects. The obtained
electron-transfer resistance can be used to determine the pin-
holes that correspond simply to the ratio of the electron-transfer
resistance of the bare electrode and the SAMs-coated electrode
[27]. The SAMs obtained using direct application method had a
significantly lower amount of pinholes by approximately two to
three times than the corresponding self-assembly immobilized
on gold by immersion method. The lower pinholes elicited that
direct application method was more effective immobilization in
the preparation of densely packed SAMs as compared to the
immersion method. Therefore, to achieve a defect-free structure
and/or close packing, the direct application method provided a
more effective, rapid, and facile self-assembly immobilization
than immersion method, consistent with the previous study [19],
attributing to the effect of gravitational force and enhanced van
der Waals forces [28].
In a study by Walczak et al. [12], it is proven that a monolayer
can be reversibly adsorbed and desorbed by electrochemical
Fig. 5. Cyclic voltammetry of 0.5 M NaOH solution (pH 13) taken with a bare
means. This process can serve as a basis for the determination of
gold electrode (a) and with gold electrodes modified with 11-MUA using immer-
the monolayer surface coverage Γ (Γ = Q/nFA), by measuring sion method (b) and direct application method (c). The scan rate is 100 mV s−1
the charge (Q). Fig. 5 shows cyclic voltammetry of 0.5 M NaOH in the potential ranging from −1.4 to −0.2 V.
50 S.-J. Ding et al. / Analytica Chimica Acta 554 (2005) 43–51
Fig. 6. Cyclic voltammograms of Au electrodes at different scan rates of:
20 mV s−1 (a), 50 mV s−1 (b), 100 mV s−1 (c), 200 mV s−1 (d), and 300 mV s−1
(e) in the potential ranging from −0.2 to +0.6 V. The supporting electrolyte is
0.1 M KCl containing 5 mM Fe(CN)6 4− .
derivatives was proportional to the square root of the potential
scan rate [29]. Based on Randles–Sevcik equation, the electroac-
tive surface area of bare Au electrode can be easily calculated
by using anodic peak current (Ipa ). It is essentially found that
the amount of ferrocyanide oxidation current is dependent of
the scan rate (Fig. 6), but electroactive area of the gold electrode
(Fig. 7). Since the different scan rates used within the poten-
tial window did not significantly (p > 0.05) result in the change
of the electroactive surface area, the area of 3.36 ± 0.03 mm2
at a scan rate of 100 mV s−1 was taken as a measure for the
microscopic surface area and, thus, as a factor for the calcu-
lation of surface coverage. Finally, we could obtain surface
Fig. 7. The relationship of electroactive surface area of the bare gold electrodes
and scan rate from 20 up to 300 mV s−1 . The electroactive surface area of the
bare Au electrode was obtained according to Randles–Sevcik equation.
coverage of SAMs on the gold electrode utilizing electroactive
surface area and charge using Γ = Q/nFA. For example, the Γ
value of 7.09 × 10−10 mol cm−2 for 11-MUA-coated electrode
by direct application method was three times greater than that
of (2.17 × 10−10 mol cm−2 ) obtained by immersion method, in
agreement with the results of Ret value. Although SAMs were
produced in time intervals as long as 60 min by using immersion
method higher than direct application method of 20 min, most
of the assembly was lost from the Au electrode surface partly
due to the cleaning process. The assembly via the preparation
of direct application method was more stable and dense on Au
electrode even during cleaning and thus it showed a higher Γ
value.
4. Conclusions
Alkanethiolate self-assembled monolayers have attracted
considerable attention because such monolayers can be tai-
lored with different chain lengths for immobilization pur-
poses. The present electrochemical measurements showed that
Fe(CN)6 3−/4− redox reactions on the gold surface were signif-
icantly blocked due to formation of SAMs. The changes in the
increased electron-transfer resistance and decreased capacitance
were more apparent at longer chain SAMs-coated electrodes
than those obtained at shorter chain SAMs by means of equiva-
lent circuit model with a CPE. The CPE capacitances of SAMs
seemed to be independent on immobilization methods. However,
the obtained Ret did provide a distinct discrimination between
direct application and immersion methods. In contrast to immer-
sion method, the direct application method may be a more effec-
tive self-assembled immobilization strategy making an attempt
of formation of densely packed self-assemblies.
Acknowledgements
The authors would like to thank the Center for Micro/Nano
Technology Research and the Center for Bioscience and
Biotechnology, National Cheng Rung University, Tainan, Tai-
wan, for equipment access and technical support. Finance
supported by the NSC fund (93-2213-E006-039) is gratefully
acknowledged.
References
[1] F. Malem, D. Mandler, Anal. Chem. 65 (1993) 37.
[2] A. Ulman, Chem. Rev. 96 (1996) 1533.
[3] M.D. Porter, T.B. Bright, D.L. Allara, C.E.D. Chidsey, J. Am. Chem.
Soc. 109 (1987) 3559.
[4] M. Collison, E.F. Bowden, M.J. Tarlov, Langmuir 8 (1992) 1247.
[5] C.M.A. Brett, S. Kresak, T. Hianik, A.M.O. Bretta, Electroanalysis 15
(2003) 557.
[6] N.K. Chaki, K. Vijayamohanan, Biosens. Bioelectron. 17 (2002) 1.
[7] T. Wink, S.J. van Zuilen, A. Bult, W.P. van Bennekom, Analyst 122
(1997) 43R.
[8] J. Anzai, B. Guo, T. Osa, Bioelectrochem. Bioenergy 40 (1996) 35.
[9] D.W. Pang, Z.L. Zhang, Y.P. Qi, J.K. Cheng, Z.Y. Liu, J. Electroanal.
Chem. 403 (1996) 183.
[10] B.G. Keselowsky, D.M. Collard, A.J. Garcia, J. Biomed. Mater. Res.
66A (2003) 247.
 S.-J. Ding et al. / Analytica Chimica Acta 554 (2005) 43–51
[11] K.B. McClary, T. Ugarova, D.W. Grainger, J. Biomed. Mater. Res. 50
(2000) 428.
[12] M.M. Walczak, D.D. Popenoe, R.S. Deinhammer, B.D. Lamp, C. Chung,
M.D. Porter, Langmuir 7 (1991) 2687.
[13] A. Badia, S. Arnold, V. Scheumann, M. Zizlsperger, J. Mack, G. Jung,
W. Knoll, Sens. Actuators B 54 (1999) 145.
[14] L. Qingwen, G. Hong, W. Yiming, L. Guoan, M. Jie, Electroanalysis
13 (2001) 1342.
[15] E. Katz, I. Willner, Electroanalysis 15 (2003) 913–947.
[16] R. Pei, Z. Cheng, E. Wang, X. Yang, Biosens. Bioelectron. 16 (2001)
355.
[17] Y.T. Long, C.Z. Li, T.C. Suther, H.B. Kraatz, J.S. Lee, Anal. Chem. 76
(2004) 4059.
[18] S.J. Ding, B.W. Chang, C.C. Wu, M.R. Lai, H.C. Chang, Electrochim.
Acta 50 (2005) 3660.
[19] M. Akram, M.C. Stuart, D.K.Y. Wong, Anal. Chim. Acta 504 (2004)
243.
51
[20] C.H. Hamann, A. Hamnett, W. Vielstich, Electrochemistry, Wiley/VCH,
Weinheim, Germany, 1998.
[21] R.V. Bucur, A. Bartes, V. Mecea, Electrochim. Acta 23 (1977)
641.
[22] J. Xu, H.L. Li, Y. Zhang, J. Phys. Chem. 97 (1993) 11497.
[23] P. Diao, M. Guo, R. Tong, J. Electroanal. Chem. 495 (2001)
98.
[24] A. Norlin, J. Pan, C. Leygraf, Biomol. Eng. 19 (2002) 67.
[25] F. Patolsky, M. Zayats, E. Katz, I. Willner, Anal. Chem. 71 (1999)
3171.
[26] GK. Jennings, J.C. Munro, T.-H. Yong, P.E. Laibinis, Langmuir 14
(1998) 6130.
[27] K.H.W. Seah, R. Thampuran, S.H. Teoh, Corros. Sci. 40 (1998) 547.
[28] P. Fenter, F. Schreiber, L. Berman, G. Scoles, P. Eisenberger, M. Bedzyk,
Surf. Sci. 412/413 (1998) 213.
[29] J. Kulys, K. Krikstopaitis, F.W. Scheller, U. Wollenberger, Electroanal-
ysis 16 (2004) 183.