J Solid State Electrochem
DOI 10.1007/s10008-016-3199-2
ORIGINAL PAPER
Enzyme-free impedimetric glucose sensor based on gold
nanoparticles/polyaniline composite film
A. J. Saleh Ahammad 1 & Abdullah Al Mamun 1 & Tania Akter 1 & M. A. Mamun 1 &
S. Faraezi 1 & F. Z. Monira 1
Received: 7 January 2016 / Revised: 20 March 2016 / Accepted: 27 March 2016
# Springer-Verlag Berlin Heidelberg 2016
Abstract A non-enzymatic impedimetric glucose sensor was
fabricated based on the adsorption of gold nanoparticles
(GNPs) onto conductive polyaniline (PANI)-modified
glassy carbon electrode (GCE). The modified electrode
(GCE/PANI/GNPs) was characterized by using cyclic
voltammetry (CV), electrochemical impedance spectros-
copy (EIS), and scanning electron microscopy (SEM).
The determination of glucose concentration was based
on the measurement of EIS with the mediation of
electron transfer by ferricyanide ([Fe(CN) 6 ] 3− ). The
[Fe(CN)6]3− is reduced to ferrocyanide ([Fe(CN)6]4−),
which in turn is oxidized at GCE/PANI/GNPs. An in-
crease in the glucose concentration results in an increase
in the diffusion current density of the [Fe(CN)6]4− oxi-
dation, which corresponds to a decrease in the faradaic
charge transfer resistance (Rct). A wide linear concentra-
tion range from 0.3 to 10 mM with a lower detection
limit of 0.1 mM for glucose was obtained. The pro-
posed sensor shows high sensitivity, good reproducibil-
ity, and stability. In addition, the sensor exhibits no
interference from common interfering substances such
as ascorbic acid, acetaminophen, and uric acid.
Keywords Glucose sensor . Gold nanoparticles .
Polyaniline . Electrochemical impedance spectroscopy
* A. J. Saleh Ahammad
ajsahammad@chem.jnu.ac.bd
1
Department of Chemistry, Jagannath University, Dhaka 1100,
Bangladesh
Introduction
Continuous monitoring of glucose level plays an important
role in clinical diagnoses, biochemical analysis, food process-
ing, and fermentation [1–3], which demands the development
of a reliable, sensitive, selective, and interference-free method.
Electrochemical methods have become the main technique in
glucose sensing because of their good sensitivity, selectivity,
and simplicity [4].
For the electrochemical sensor applications, the electrode-
modifying material is expected to possess several characteris-
tics such as good electron transduction capability, easy acces-
sibility toward the analyte, and large surface area.
Unfortunately, all these important characteristics cannot be
built-in in a single material. Hence, there is always a demand
for the development of composite materials to achieve
adequate sensitivity and stability for the electrochemical
sensors [5–7]. Among various composite materials,
conducting polymer-metal nanoparticles, composite films
have received considerable attention because of their unique
physical and chemical properties and various applications in
many research areas [8].
Polyaniline (PANI) is the most widely investigated
conducting polymer that have properties of high conductivity,
good redox reversibility, and stability [9]. It has been used in
many practical applications, such as electrocatalysis [10],
chemosensors [11], and electrochemical sensors [12]. On the
other hand, gold nanoparticles (GNPs) have been extensively
studied in electroanalytical chemistry because of its unique
properties such as easy to synthesize, good biocompatibility,
high electrocatalytic activity, and large surface-to-volume ra-
tio [13, 14]. Moreover, GNPs exhibit fascinating properties for
the oxidation of glucose, such as stable chemical property,
good conductivity, and anti-poisoning property [15]. Taking
into account the good performance of PANI and GNPs, an

advanced composite material (GNP-PANI film) was prepared
in this study.
The GNP-PANI composite films are mainly used for
the fabrication of enzyme-based amperometric glucose
sensor. A. D. Chowdhury et al. [16] fabricated an am-
perometric glucose-sensing platform by immobilizing
GOx onto PANI decorated with GNPs. V. Mazeiko
et al. [8] developed an amperometric glucose sensor
by entrapping GOx and GNPs within PANI layer. W.
Hou and co-workers [17] entrapped GOx only onto
the nanocomposite with a core-shell structure containing
GNPs, PANI, and polystyrene (PS) for the construction
of amperometric glucose sensor.
Nevertheless, one severe problem of enzyme-based glu-
cose sensors is their lack of stability due to the intrinsic nature
of enzymes [18, 19]. Activity of enzyme can be seriously
affected by temperature, pH, oxygen, humidity, toxic
chemicals, and so on. Moreover, a complicated procedure,
including functionalization, cross-linking, adsorption, or en-
trapment, is required for the immobilization of the enzyme on
the electrode surfaces [20]. In order to overcome these draw-
backs, we have concentrated on the development of non-
enzymatic glucose sensor, which is suitable for practical
applications.
The amperometric biosensors are based on measure-
ment of the current resulting from electrochemical ox-
idation or reduction of an electroactive species.
However, they have their own inherent limitations,
such as noisy response and overvoltage applied to the
biosensor [21]. Therefore, it is necessary to look for a
new electrochemical transducers based on other
methods such as electrochemical impedance spectrosco-
py (EIS).
The EIS is a powerful and non-destructive technique,
which is usually used for characterization and study of
corrosion phenomena [22], fuel cells and batteries [23],
coatings and conductive polymers [24], adsorption be-
havior of thin films [25], and electron transfer kinetics
[26]. R. K. Shervedani and co-workers [21] used EIS to
determine glucose in the presence of parabenzoquinone
(PBQ) mediator based on the immobilization GOx en-
zyme on the topside of the gold mercaptopropionic acid
self-assembled monolayers.
In this study, we have prepared the enzyme-free sensor by
attaching GNPs with the PANI-modified glassy carbon
electrode (GCE). Next, the sensor is used to determine glucose
in the presence of [Fe(CN)6]3− mediator using EIS. The basis
of the recognition system in this work is diffusion of glucose
to the sensor. Easy preparation of GNP-PANI composite,
appreciable electrical conductivity of the electrode surface,
and strong attachment between GNPs and PANI improves
overall stability of the sensor and results in much efficient
sensing process.
J Solid State Electrochem
Experiment
All reagents were obtained as analytical grade and used with-
out further purification. Double-distilled water was used
throughout the experiments. Glucose, polyaniline, gold nano-
particles (10 nm, 0.01 % HAuCl4), potassium ferricyanide,
sodium phosphate dibasic, and sodium phosphate monobasic
were purchased from Sigma-Aldrich (Germany). Phosphate
buffer solution (PBS) was prepared following our previous
procedure [27]. All experiments were performed at room
temperature.
PANI-modified GCE was prepared by using drop-casting
method. At first, PANI solution was prepared in a mixture of
acetone and ethyl acetate (2:3 v/v). The solution was then
dropped onto GCE, which was polished with 0.05-μm
alumina/water slurry (Buehler, USA) on a polishing cloth to
a mirror-like finish, followed by sonication, and then rinsed
with distilled water. After that, the solution was dried on air.
The resulting PANI-modified GCE (GCE/PANI) was rinsed
with PBS and afterward dipped into a GNP solution for 6 h at
4 °C. Finally, the modified electrode (GCE/PANI/GNPs) was
washed with PBS (pH 7.0) and stored in a refrigerator (4 °C)
when not in use.
Electrochemical experiments were performed with a CHI
660 E electrochemical workstation (CH Instruments, USA). A
conventional three-electrode system was used where GCE or
modified GCE electrode, a platinum wire, and a Ag/AgCl
(3 M KCl) electrode were used as working, counter, and ref-
erence electrodes, respectively. All potentials in this study
referred to this reference electrode. EIS measurements were
carried out in a three-electrode cell with a 5 mM K3[Fe(CN)6]
solution containing with 1 M KCl/0.1 M PBS. Impedance
spectra were recorded over a frequency range from 100 kHz
to 1 Hz with amplitude of 5 mV using CHI 660 E. The mea-
surements were performed at the formal redox potential of
[Fe(CN)6]3−, 0.3 V, and the measured spectra were fitted to
an appropriate equivalent circuit on Zview software (version
3.1, Scribner Associates Inc., USA). A field emission scan-
ning electron microscope (FE-SEM; Jeol, model JSM 7600 F)
was used to investigate the morphology of the modified
electrodes.
Results and discussion
Figure 1 shows the schematic diagram for the fabrication of
glucose sensor. At first, PANI-modified GCE is prepared via a
drop-casting process. The polymer-coated electrode (GCE/
PANI) was immersed in a GNP solution for 6 h, at 4 °C.
GNPs were attached onto the PANI film through the strong
interaction between negatively charged GNPs and the posi-
tively charged functional group NH2 of PANI [14]. The
adsorbed GNPs were observed via SEM analysis. Figure 2
J Solid State Electrochem
Fig. 1 Schematic diagram for the
fabrication of glucose sensor
based on the impedimetric
measurement
shows the SEM images of the GCE/PANI- and GCE/PANI/
GNP-modified electrode surfaces. As can be seen, no nano-
particles were found on the GCE/PANI (Fig. 2a). On the other
hand, well distribution of GNPs onto the PANI film was ob-
served, as shown in Fig. 2b. Almost no aggregation of the
nanoparticles on the surface was found. This indicates that
the GNPs were successfully attached onto the PANI-
modified GCE. The GNPs on the PANI film were quite stable
and could withstand repeated rinsing with water.
The surface properties of the modified electrodes were
characterized with cyclic voltammetries (CVs). Figure 3
shows the CV responses of 5 mM [Fe(CN)6]3− in 1 M KCl
solution at GCE, GCE/PANI, and GCE/PANI/GNPs. At bare
GCE, a well-defined redox wave of [Fe(CN)6]3− is observed
(Fig. 2a) as reported previously [28]. After the deposition of
PANI onto GCE, the CV curve changes significantly from an
electrochemically reversible shape to a quasi-reversible shape
(Fig. 2b) due to the sluggish electron-transfer kinetics through
the PANI film. However, a better redox wave of [Fe(CN)6]3−
was obtained after the adsorption of GNPs onto PANI
(Fig. 3c). Obviously, the GNPs on PANI film have influence
on the interfacial properties of the modified electrode and
improve the electron transfer between [Fe(CN)6]3− and the
electrode. However, the reversible shape is not restored due
to the repulsion of negatively charged GNPs [29] with the
negatively charged ferricyanide.
Fig. 2 SEM images of the a
GCE/PANI and b GCE/PANI/
GNP
The modified electrodes were further characterized
with EIS. Figure 4 shows the Nyquist plots of the EIS
of the bare and modified electrodes. The semicircle por-
tion at the higher frequencies corresponds to the
electron-transfer limited process, and the diameter is
equivalent to the electron transfer resistance (Rct). The
linear part at lower frequency corresponds to the
diffusion-controlled process. At a bare GCE, the redox
process of the [Fe(CN)6 ] 3− probe showed an R ct of
about 900 Ω. The GCE/PANI showed a higher resis-
tance for the redox probe, while the resistance decreased
with the GCE/PANI/GNP electrode. This indicates that
the GNP-modified electrode possesses good conductive
interface. Overall, the impedance results were quite con-
sistent with the conclusion from the CV experiments
and demonstrate that PANI and GNPs are successfully
attached on the GCE.
For evaluating the electrochemical properties of the
GNPs on PANI, CVs of the GNP-modified electrode
were carried out in 0.1 M PBS (pH 7) as shown in
Fig. 5. No faradic current was observed on the GCE/
PANI. On the other hand, the characteristic oxidation
and subsequent reduction peaks of gold were clearly
observed on the GCE/PANI/GNPs. The typical anodic
oxidation of the gold took place at a potential of
1.39 V, while the cathodic reduction peaks that appeared
 J Solid State Electrochem

Fig. 3 CVs of 5.0 mM K3[Fe(CN)6] in 1 M KCl at GCE (a), GCE/PANI

(b), and GCE/PANI/GNP (c). Scan rate = 100 mV/s Fig. 5 CVs of 0.1 M PBS (pH 7.0) at GCE/PANI (a) and GCE/PANI/
GNP (b) with a potential scan rate of 100 mV/s

at 0.33 V were attributed to the formation of gold oxide

and subsequently its reductive reaction, respectively. glucose is oxidized to gluconolactone. The reduced
This confirms that the redox peaks for the GCE/PANI/ [Fe(CN)6]3− is electrochemically oxidized on the elec-
GNPs came from the GNPs. trode as the redox reaction ([Fe(CN)6]3−/[Fe(CN)6]4−)
To verify the possibility of developing a glucose biosensor is fast and reversible. Therefore, the extent of
with GCE/PANI/GNPs, the characteristics of the glucose [Fe(CN)6]4− formation is controlled by the concentration
charge transfer reaction were studied by means of EIS. of glucose and [S] can be replaced with k1 [glucose],
Figure 6a shows the EIS data recorded for various concentra-
tions of glucose. It is clear from the figure that the electron
transfer resistance is decreased as the concentration of glucose
increased. This can be explained by the equation [21]
Rct = RT/(n2F2Akct[S]), where R is the ideal gas constant, T
is the absolute temperature, n is the number of transferred
electrons per one molecule of the redox probe, F is the
Faraday constant, A is the geometric surface area of the elec-
trode (cm2), kct is the potential dependent charge transfer rate
constant, and [S] corresponds to the concentration of the redox
probe (mol/cm 3 ). The GCE/PANI/GNP electrode was
examined as a catalytic interface to produce [Fe(CN)6]4− from
[Fe(CN)6]3− using faradaic impedance spectroscopy. The elec-
trocatalytic oxidation of glucose onto GNPs has been reported
earlier [30]. The [Fe(CN)6]3− is reduced to [Fe(CN)6]4−, while

Fig. 6 a EIS responses as a function of concentration of glucose (a–i 0.0,

Fig. 4 EIS plots of 5.0 mM K3[Fe(CN)6] in 1 M KCl at GCE (a), GCE/
PANI (b), and GCE/PANI/GNP (c). The frequency range was from 0.1 to
100,000 Hz with perturbation amplitude of 5 mV
0.3, 0.5, 0.7, 1.0, 2.0, 3.5, 7.0, and 10.0 mM) in 0.1 M PBS (pH 7.0)
containing 5.0 mM K3[Fe(CN)6] at GCE/PANI/GNP. Inset shows an
equivalent circuit model and the magnification of EIS data at high-
frequency region. b Corresponding calibration plot of 1/Rct vs. glucose
concentration
J Solid State Electrochem

Table 1 Comparison of the

proposed glucose sensor with the Modified electrode Linear range (mM) Detection limit (mM) Reference
others based on GNPs or PANI-
modified electrode GCE/PANI/GNPs/Cyt c/GOD 0.01–3.2 0.01 [32]
GE /PANI/GOD 1–5 – [33]
Au/DT/GNPs/CT/GOD 0.02–5.7 0.01 [34]
GCE/GNPs/PPyNFs 0.2–13 – [35]
Pt/POT/GNPs/MPB 5–30 0.2 [36]
GCE/GNPs/GOD/CNTs/PV 0.5–8 0.2 [37]
GCE/OOPPy/GNPs/GOD 1–8 0.5 [38]
GCE/Pt HNPCs/GNPs/CS 3–7.7 1.0 [39]
GCE/PANI/GNPs 0.3–10 0.1 This work

GOD glucose oxidase, Cyt c cytochrome c, GE graphite electrode, DT dithiol, CT cystamine, PPyNFs polypyrrole
nanofibers, POT poly(3-octylthiophene), MPB 4-mercaptophenyl boronic acid, PV polyvinyl alcohol, OOPPy
overoxidized polypyrrole, Pt HNPCs platinum hollow nanoparticles

where k1 is a constant. A linear relation as 1/Rct = k
[glucose] is simply found, if all other parameters are
also constant. Therefore, the values of the charge trans-
fer resistances gradually decrease with the increase of
glucose concentration. Figure 6b contains the calibration
curve calculated as the inverse of charge transfer resis-
tance values vs. glucose concentrations. For this curve,
the linearity range is 0.3–10 mM and a LOD (limit of
detection) = 0.1 mM is defined following the previous
report [31]. The linear range and detection limit were
comparable with other GNP- or PANI-based glucose
sensors as shown in Table 1.
The reproducibility of GCE/PANI/GNP electrode was
determined by 20 successive impedimetric measure-
ments toward 1 mM glucose with the same modified
electrode. The relative standard deviation (RSD) of the
measurements was 2.5 %. The stability of the electrode
was also investigated. After the GCE/PANI/GNP elec-
trode was stored in refrigerator for 2 weeks, the
impedimetric response of 1 mM glucose was stable
and maintained 95 % compared to its original value.
The sensor was then tested by EIS in the presence of
glucose and the three interferents in order to verify the
selectivity of the proposed sensor. Figure 7 shows the
Nyquist plots of glucose in the presence of ascorbic
acid (AA), uric acid (UA), acetaminophene (AP), D-
galactose (GT), and L-tyrosine (TS). In fact, the only
semicircle present is due to the glucose reaction, which
confirms the effective selectivity of the biosensor. This
biosensor has demonstrated the possibility of using EIS
as efficient technique for the detection of molecules of
biological interest. Work is in progress in our laboratory
to improve this result applying this method to real
samples.
Conclusions
We have shown that EIS can be used as a basis for the
determination of glucose. Since the DC potentials ap-
plied in the EIS measurements (e.g., +0.3 V) are less
anodic than that applied in amperometric measurements
(e.g., +0.6 V), more accurate data can be obtained in
the EIS method. Thus, the inherent problems due to
high DC overvoltage applied in amperometric measure-
ments can be overcome in impedimetric method. A wide
linear range and a low limit of detection were obtained
for the determination of glucose. The proposed glucose
sensor exhibited good stability, reproducibility, and
selectivity.

Acknowledgments This work was supported by The World Academy

Fig. 7 EIS plots of glucose (1.0 mM) in 0.1 M PBS (pH 7.0) containing of Sciences (TWAS) Research Grant Program (Ref. 12–183 RG/CHE/
5.0 mM K 3 [Fe(CN) 6 ] at GCE/PANI/GNP without (a) and with AS_I and UNESCO FR 12–183 RG/CHE/AS_I). We thank Dr. Subrata
interferences (0.1 mM; b ascorbic acid, c uric acid, d acetaminophene, Sarker at Hongik University for valuable discussion regarding electro-
e D-galactose, and f L-tyrosine) chemical impedance spectroscopy.

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