Electrochemistry Communications 33 (2013) 127–130

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Electrochemistry Communications
journal homepage: www.elsevier.com/locate/elecom

Short communication

Label free MUC1 aptasensors based on electrodeposition of gold

nanoparticles on screen printed electrodes
Anca Florea a, Zahra Taleat a,b, Cecilia Cristea a, Mohammad Mazloum-Ardakani b, Robert Săndulescu a,⁎
a
Analytical Chemistry Department, Faculty of Pharmacy, Iuliu Haţieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania
b
Department of Chemistry, Faculty of Science, Yazd University, Yazd, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Two simple electrochemical aptasensors were developed for the detection of MUC1 tumor marker based on
Received 19 April 2013 its specific recognition by thiolated aptamers immobilized on gold nanoparticle (Au NP)-modified graphite
Received in revised form 10 May 2013 and gold screen printed electrodes (SPEs). The quantitative detection of MUC1 protein was achieved by electro-
Accepted 10 May 2013
chemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). Calibration curves were
Available online 16 May 2013
obtained with both techniques under optimized conditions. The estimated detection limits of the MUC1 protein
Keywords:
are 3.6 ng mL−1 at Au NP-modified graphite SPE by EIS and 0.95 ng mL−1 at Au NP-modified gold SPE by DPV
Gold nanoparticles methods. The results demonstrated that the electrochemical method using aptamer probe immobilized on Au
MUC1 NPs is convenient and allows the quantitative detection of MUC1 protein.
Aptamer © 2013 Elsevier B.V. All rights reserved.
Screen printed electrode

1. Introduction cells/mL [17–19]. An electrochemical sensor for MUC1 was developed

Detection of tumor markers plays an important role in clinical diagno-
sis and evaluation of treatment for patients with certain tumor-associated
diseases [1]. The drawbacks of the conventional techniques for the detec-
tion of tumor markers in serum lead to the requirement for the urgent
development of new methods with low-cost, high speed and real-time
control in large-scale disease screening [2]. Immunosensors combine
the advantages of sensors with high sensitivity and the immunoreactions
with high specificity, many immunoassays being developed for the detec-
tion of tumor markers [3–8].
MUCl, a transmembrane glycoprotein expressed by most ‘wet’ epi-
thelia [9], is overexpressed and shed into blood circulation in case of a
malignant process, where it can be measured by various immunoassays
[10].
Aptamers may provide a potential solution for early diagnosis of
cancer [11]. Aptamers are synthetic nucleic acid ligands (DNA or RNA)
which can be selected from combinatorial libraries of synthetic nucleic
acids, to have specific affinity for cancer biomarkers.
Aptamer based detection methods are attractive due to their high
sensitivity, selectivity to the target molecule and good stability in
complex environments. A number of aptasensors for small molecules,
proteins, even cells have been developed [12,13] and used for the
construction of biosensing devices [14–16]. Recently, based on the
discovery of DNA aptamers targeting MUC1, a few aptamer-based
sensors have been developed to detect MUC1-overexpressed breast
cancer cells with detection limits between 104–107 and 30 cancer
using ferrocene carboxylic-doped silica nanoparticles as an immobili-
zation support, with a detection limit of 0.64 U/mL [20].
The key issues with any electrochemical aptamer-based biosensor
include the enhancement of aptamer immobilization amount, mainte-
nance of target accessibility and the demonstration of useful detection
signals [10]. Gold nanoparticles (Au NPs) are excellent candidates for
bioconjugation, owing to the fact that they are biocompatible and
bind readily to a range of biomolecules. The immobilization of the
nanoparticles on the electrode increases the surface area and promotes
the adsorption capability of the electrode.
Hence, in this work, we have developed two electrochemical assays
based on a MUC1-binding aptamer immobilized on graphite and gold
screen printed electrodes (SPE) modified with Au NPs. The loosely
packed aptamers are self-assembled onto gold surface. In impedance
measurements, an increase in charge transfer resistance is observed
after immobilization of proteins to the sensor surface. In the second ap-
proach, the electrochemical response of methylene blue (MB) is studied
in the presence of different concentrations of MUC1 protein. Binding of
the MUC1 protein to the aptamer prevents the subsequent intercalation
of MB. Therefore, the electrochemical response is lower than that ob-
served in the absence of protein. The parameters that could affect the
sensor response were optimized.
2. Experimental
2.1. Chemicals and instrumentation

⁎ Corresponding author. Tel.: +40 264597256x2412; fax: +40 264597257. MUC1 protein was provided by Novus Biological. Aptamer 5′-GCA
E-mail address: rsandulescu@umfcluj.ro (R. Săndulescu). GTTGATCCTTTGGATACCCTGGTTTTTTTTTTTTTTT-3′-SH was purchased

1388-2481/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.elecom.2013.05.008
128 A. Florea et al. / Electrochemistry Communications 33 (2013) 127–130
from AlphaDNA (Canada). Na2HPO4, NaH2PO4, K4[Fe(CN)6]), K3
[Fe(CN)6], H2SO4, methylene blue (MB) and Tris (hydroxymethyl)
aminomethane (TRIS) were purchased from Merck. 6-Mercapto-
1-hexanol (MCH), NaCl, MgCl2, HAuCl4 were purchased from
Sigma-Aldrich. All solutions were prepared using MilliQ water. The
following buffers were used: phosphate buffered saline pH 7.4
(buffer A) and Tris 10 mM, NaCl 100 mM, KCl 100 mM and MgCl2
5 mM pH 7.2 (buffer B).
Graphite SPEs with graphite working electrode, graphite counter
electrode and a silver pseudo-reference electrode and gold SPEs with
gold working electrode, gold counter electrode and a silver pseudo-
reference electrode were purchased from Dropsens (Spain).
Electrochemical measurements were performed with AUTOLAB
PGSTAT 30 (EcoChemie, Netherlands) with GPES and FRA2 software
4.9.
EIS measurements were carried out in the presence of 10 mM
[Fe(CN)6]3/4− in buffer A, in a frequency range from 100 mHz to
100 kHz at a dc potential of +0.13 V. The data is represented in
Nyquist plots.
DPV was recorded within the potential range of − 0.6 to 0 V under
modulation amplitude of 70 mV and a scan rate of 50 mV s−1.
Cyclic voltammetry (CV) was employed for the electrodeposition
of Au NPs on SPE in the potential range of − 0.2 to 1.2 V, as well as
for the detection of MUC1 antigen in the potential range of − 0.6 to
0 V, with a scan rate of 100 mV s−1.
The microscopic images were obtained with a WITEC Alpha 300
System.
2.2. Preparation of Au NP-modified SPE
To construct the two aptasensors, the surface of the working elec-
trode was modified with Au NPs by electrodeposition using a solution
of HAuCl4 0.6 M in H2SO4 0.5 M and cycling the potential between
−0.2 and 1.2 V, with a scan rate of 100 mV s− 1, for 15 cycles.
Prior to electrodeposition the gold SPEs were electrochemically
cleaned with 0.5 M H2SO4 by potential scanning within a range of
− 0.2 to 1.2 V.
2.3. Immobilization of the aptamer onto Au NP-SPE surface and interaction
with protein
For the graphite-Au NP-based sensor 10 μL of 5 μM aptamer solu-
tion was placed on the working electrode and incubated for 30 min,
followed by an incubation step with 10 μL of MUC1 solution in buffer
B for 60 min.
In case of gold SPE-Au NP-based sensor, the self assembled mono-
layer was spontaneously formed through incubation overnight in a
solution of 10 μM aptamer at 4 °C, followed by the reaction with
1 mM MCH solution for 1 h to mask the unreacted Au sites. MB was
accumulated onto the modified SPE by adding 1 mM MB solution
for 15 min, proceeding afterwards with the reaction with different
concentrations of MUC1 solution for 90 min. The electrode was rinsed
with doubly distilled water after each incubation step.
2.4. Optimization of the assay
2.4.1. Optimization of experimental conditions of Au NP-graphite SPE
aptasensor by EIS
The efficient immobilization of the thiolated aptamer on Au NPs is
of great importance for the successful development of the aptasensor.
Different concentrations of aptamer solution and different incubation
times were assessed in order to achieve a high coverage of the elec-
trode surface with aptamers for sensitive antigen detection. Keeping
the aptamer concentration fixed to 1 μM, the reaction time with the
aptamer was varied between 5, 10, 30 and 60 min, leading to an in-
crease in the charge transfer resistance as the time was increased
from 5 to 30 min showing an increasing immobilization of aptamer
with time on the Au NPs. Increasing the reaction time to 60 min
resulted in a relatively small increase in the signal, therefore an incuba-
tion time of 30 min was considered optimal. The charge transfer resis-
tance also increased with an increase of the aptamer concentration up
to 5 μM when the signal tends to level off, indicating a saturation of
the Au NP-modified electrode surface with aptamers. The sensor was
tested for a concentration of aptamer of 10 nM, 100 nM, 1 μM, 5 μM
and 10 μM. The incubation time with the antigen was further optimized
incubating the aptamer/Au NP-modified SPE with 10 ng mL−1 MUC1
solution for 15, 30, 60 and 90 min. The signal increases with the in-
crease of the incubation time up to 60 min.
2.4.2. Optimization of experimental conditions of Au NP-gold SPE
aptasensor by DPV
The incubation time of aptamer on the electrode was optimized, in-
cubating the Au NP-modified electrode with aptamer solution for 2 h
and overnight. The current ratio between signal and blank (I5 ng/mL/I0)
increased from 1.4 to 1.9 as the incubation time increased from 2 h to
overnight. A longer incubation time was required in this case due to a
larger gold surface available for the binding of thiolated aptamer, thus
overnight incubation was selected as the adequate incubation time for
the aptamer.
3. Results and discussion
3.1. Principle of electrochemical aptamer-based biosensor
Fig. 1A illustrates the principle of our two different approaches for
the detection of MUC1 based on its specific recognition by thiolated
aptamers immobilized on Au NP-modified SPEs.
3.2. Characterization of Au NP-graphite SPE-based aptasensor
The graphite SPEs were modified with Au NPs as immobilization
platform for the thiolated aptamers and the features of the sensor
were investigated by EIS, using 10 mM [Fe(CN)6]−3/−4 in buffer A
as a redox probe. The electrodeposition of Au NPs lead to a significant
decrease in the charge transfer resistance (a) with respect to the bare
graphite electrode (b), improving the electron transfer at the elec-
trode surface as shown in Fig. 2A. The immobilization of the aptamer
on the Au NP-modified surface gave rise to an increase in the electron
transfer resistance (Rct) (c) as a consequence of the electrostatic re-
pulsion between [Fe(CN)6]−3/−4 anions and the negatively charged
aptamers, which indicates that aptamers have been immobilized on
SPEs obstructing the electron-transfer. After incubation with MUC1
protein, the interfacial resistance increased more due to the interac-
tion of aptamer with MUC1 protein, a large protein with a molecular
weight of 250–1000 kDa, bringing a steric hindrance effect on the
electron transfer of [Fe(CN)6]−3/−4 through the electrode (d).
Microscopy was used to confirm the electrodeposition of Au NPs on
the SPE surface. The microscopy images show a random distribution of
the gold particles on the electrode surface, as well as pores and holes
(Fig. 2B). The results are in good agreement with the SEM images
reported by Ravalli et al. [21].
For the gold SPE based sensor, CV and DPV were applied to monitor
the process of fabrication of the aptasensor after each step (data not
shown). Measurements were carried out using buffer A for the various
modified electrodes incubated with 1 mM MB solution. The bare SPE
exhibited small peak currents confirming that MB binds negligibly to
the electrode surface, thus the electrochemical signal of the cyclic
voltammograms mainly resulted from the intercalated MB. After the
immobilization of aptamers, the current increased due to the accumula-
tion of MB onto the aptamer-modified SPE, based on its specific interac-
tion with the guanine bases of the aptamer [22]. After incubations with
different concentrations of MUC1 protein, it can be clearly seen that the
 A. Florea et al. / Electrochemistry Communications 33 (2013) 127–130 129

Route A
-S- -S-
HS -
-S- -S-

-S- -S-
a) b) -S-
c) -S-
d)

Route B

HS -
-S- -S-

a) b)
-S-
f)
-S-
h)
e) -S- g) -S-

Gold nanoparticle HS - Thiolated aptamer

Methylene blue
Mercaptohexanol
MUC1 protein

Fig. 1. Detection of MUC1 by EIS on Au NP-graphite SPE (route A) and by DPV on Au NP-gold SPE (route B); (a) electrodeposition of Au NPs, (b) immobilization of thiolated aptamer,
(c) affinity reaction with MUC1, (d) EIS measurements, (e) reaction with MCH, (f) accumulation of MB, (g) incubation with MUC1 and (h) DPV measurements.

current decreases gradually as the concentration of MUC1 protein in-
creases, due to the reduction of the amount of intercalated MB in the
aptamer.
DPV responses show that the peak of MB was not significant on the
bare gold SPE, but a large signal for the intercalated MB was obtained
after the immobilization of aptamers. After incubation of 5 ng mL−1
MUC1 solution, the reduction signal of MB decreased. The cyclic
and differential pulse voltammograms strongly support the fact that
the aptamer was immobilized on the electrode surface and MB was
properly intercalated in the aptamer.

Fig. 2. (A) Nyquist plots of EIS data performed in 10 mM [Fe(CN)6]−3/−4 solution for: (a) graphite SPE/Au NPs, (b) bare graphite SPE (c) graphite SPE/Au NPs/Aptamer and (d) graphite
SPE/Au NPs/Aptamer/MUC1 (5 ng mL−1); (B) microscopy image of electrodeposited Au NPs/graphite SPE; (C) change of the electron-transfer resistances (Rct) at the modified gold SPEs
and (D) DPVs for the incorporated MB on aptamer modified Au NPs/gold SPE upon the analysis of different concentration of MUC1 protein; (panel C (a) 2.5, (b) 5, (c) 7.5, (d) 10 and
(e) 15 ng mL−1 MUC1 and panel D (a) 0, (b) 2, (c) 4, (d) 5, (e) 7.5, (f) 9 and (g) 10 ng mL−1 MUC1; insets: dose–response curve for different concentrations of MUC1 protein;
EIS parameters: potential +0.13 V, frequency range 100 mHz–100 kHz; DPV parameters: potential range −0.6 to 0 V, modulation amplitude 70 mV, scan rate 50 mV s−1).
130 A. Florea et al. / Electrochemistry Communications 33 (2013) 127–130
3.3. Analytical performance of the aptasensors
A dose–response curve was obtained under optimized experimental
conditions by both EIS (Fig. 2C) and DPV (Fig. 2D) techniques. A linear
response was obtained in the range of 2.5–15 ng mL−1 (y = 0.545x +
7.801, R2 = 0.968) and 0–10 ng mL−1 (y = 1.222x + 13.209, R2 =
0.981) with a limit of detection of 3.6 ng mL−1 and 0.95 ng mL−1,
respectively.
4. Conclusion
Two simple and sensitive strategies for the electrochemical detec-
tion of MUC1 protein using aptamer and Au NPs were introduced in
this work. Loosely packed aptamer were self-assembled onto SPE surface
modified with Au NPs. The interaction between aptamer and MUC1
protein was investigated by CV, EIS and DPV techniques. The results
demonstrate that the electrochemical method using aptamers im-
mobilized on Au NPs is convenient, and allows quantitative detection
of human MUC1 protein, providing promising perspectives for future
clinical applications.
Acknowledgments
The authors are grateful for the financial support to the Romanian
National Authority for Scientific Research, CNCS-UEFISCDI, project
number PN-II-ID-PCE-2011-3-0355.
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