Colloids and Surfaces A: Physicochem. Eng.

Aspects 413 (2012) 224–230

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces A: Physicochemical and

Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa

Glucose biosensor based on glucose oxidase and gold nanoparticles of different

sizes covered by polypyrrole layer
Natalija German a , Arunas Ramanavicius b,c , Jaroslav Voronovic b , Almira Ramanaviciene a,b,∗
a
Division of Immunotechnology, State Research Institute Center for Innovative Medicine, Zygimantu 9, LT-01102 Vilnius, Lithuania
b
Center of Nanotechnology and Materials Science – NanoTechnas, Faculty of Chemistry, Vilnius University, Naugarduko 24, LT-03225 Vilnius, Lithuania
c
Department of Materials Science and Electronics, Institute of Semiconductor Physics, State Scientific Research Institute Centre for Physical Sciences and Technology, Vilnius, Lithuania

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

Article history: Different glucose biosensors based on glucose oxidase (GOx) immobilised on bare carbon rod electrode
Received 31 October 2011 (CR) modified with gold nanoparticles (Au-NPs) of (i) 3.5 nm (GOx/3.5Au-NPs/CR), (ii) 6 nm (GOx/6Au-
Received in revised form 14 February 2012 NPs/CR) and (iii) 13 nm (GOx/13Au-NPs/CR) were investigated and compared with biosensors based on
Accepted 15 February 2012
GOx immobilised on bare CR (GOx/CR). Enzymatic polymerisation of pyrrole was applied to increase
Available online 3 March 2012
linear detection range of biosensors. The influence of the formed polypyrrole layer on sensitivity and
Michaelis–Menten kinetics of designed electrochemical biosensors was investigated. The linear glu-
Keywords:
cose detection interval for GOx/CR and GOx/Au-NPs/CR electrodes was dependent on the duration of
Carbon rod electrode
Electron transfer
polymerisation.
Gold nanoparticles © 2012 Elsevier B.V. All rights reserved.
Glucose oxidase
Amperometry
Biosensor

1. Introduction Electrically conducting materials based on ␲–␲ conjugated

Nanotechnology has recently become one of the most excit-
ing forefront fields offering technological advantages suitable for
analytical chemistry [1–4]. In some cases a combination of nano-
materials and nanotechnological approaches resolves challenging
bioanalytical problems, including specificity, stability and sensitiv-
ity [5,6]. Several reviews have recently appeared demonstrating the
advantages of different nanomaterials and polymers in the design
of electrochemical biosensors and immunosensors [2–4,7–10]. Au-
NPs have been used as electron-transfer mediators and electric
wires for enhancing the electron-transfer rate between the active
centre of enzymes and electrodes [11]. The practical advantage of
Au-NPs is that their size and surface morphology can be controlled
experimentally adjusting the preparation conditions [12]. It was
shown that Au-NPs increase the efficiency of enzyme immobilisa-
tion [13,14]. To exploit this unique property many electrochemical
biosensors based on gold nanoparticles were designed and applied
for biochemical, clinical and environment needs, including can-
cer diagnostics, detection of infectious microorganisms, sensing of
vitamins, amino acids, sugars, and pesticides [2,3,5,6,15,16].
∗ Corresponding author at: Division of Immunotechnology, State Research Insti-
tute Center for Innovative Medicine, Zygimantu 9, LT-01102, Vilnius, Lithuania.
Tel.: +370 67203653, fax: +370 52330987.
E-mail address: a.ramanaviciene@imcentras.lt (A. Ramanaviciene).
polymers are often used as electrocatalysts or immobilisation
matrix for biomolecules. Conjugated polymers provide effective
immobilisation patterning for biomolecules on different substrates
[17]. Moreover in some cases ␲–␲ conjugated polymers facili-
tate electron transfer from enzymes to electronically conductive
electrodes [18,19]. Most promising electrically conducting poly-
mers (e.g., polyaniline, polypyrrole (Ppy)) are chemically stabile
on different substrates [20]. Increased interest in synthesis of chi-
ral conducting polymers has recently been observed due to their
potential application such as electrochemical asymmetric synthe-
sis. Electrochemical properties of polymers (e.g., Ppy) strongly
depend on their red-ox states, and overoxidation level, which
occurs at positive potentials and leads to de-doping of anionic
molecules followed by lowering of polymer conductivity [18,19].
Conducting polymers are easily synthesised by electrochemical
[3,21–24], chemical [25,26] and enzymatic [27] oxidative polymeri-
sation techniques [18,19]. Insoluble, stable in ambient conditions
Ppy films usually are prepared electrochemically by the oxidation
of commercially available pyrrole monomers [24,28]. On the other
hand, electrochemical polymerisation methods have a limitation:
it is difficult to prepare a large amount of Ppy. In contrast, chemical
polymerisation is suitable for large-scale production of conjugated
polymers [29,30]. Chemical polymerisation occurs by oxidation of
pyrrole monomers and formed oligomers to the cation-radicals,
which recombine and form polymeric structure of polypyrrole
[18,31]. During chemical polymerisation of pyrrole using HAuCl4

0927-7757/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfa.2012.02.012
 N. German et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 224–230
as oxidizing agent the formation of a nanostructured Ppy/Au-NP
composite on the surface of the working electrode is observed [25].
The nanometer size of nanoparticles enables facilitating direct and
fast electron transfer between the red-ox species and the trans-
ducer [1,32]. On the other hand, entrapment of Au-NPs within
the Ppy film might enhance electrical conductivity of the poly-
mer. Shorter polymerisation duration (less than 30 min) resulted
in an unstable film, which could be easily desorbed from the sur-
face of the electrode. However, very long polymerisation duration
(over 200 h) resulted in a formation of thicker polymer layer fol-
lowed by a significant decrease of the analytical signal [25]. Major
disadvantages of chemical methods are limitations to be used
for the construction of miniaturised sensor arrays [18]. Usually
electrically conducting polymers serve as a matrix for the incorpo-
ration of metal particles and for the immobilisation of the enzyme,
and are excellent transducers for the design of modern elec-
trochemical (amperometic, impedimetric) biosensors [3,25,26,33],
immunosensors [9,19], DNA sensors [34], biomedical applications
in vitro [26] and in vivo [21,26,29,35]. Moreover polypyrrole is
biocompatible [36] and protects electrodes from fouling and inter-
fering with electrochemically active materials [19].
The main aim of this research was to investigate the influence
of ␲–␲ conjugated polymer, polypyrrole, layer formed on GOx/CR
and GOx/Au-NPs/CR electrodes on sensitivity, stability and lin-
ear detection range of biosensors using Au-NPs of different sizes.
Some kinetic properties and analytical characteristics of GOx/CR
and GOx/Au-NPs/CR electrodes were evaluated and compared.
2. Experimental
2.1. Chemicals
Glucose oxidase (EC 1.1.3.4, type VII, from Aspergillus niger,
215.266 units mg−1 protein) and N-methylphenazonium methyl
sulphate (PMS) were purchased from Fluka and Sigma–Aldrich
(Buchs, Switzerland), respectively. d-(+)-Glucose, tetrachloroau-
ric acid (HAuCl4 ·3H2 O) and tannic acid were obtained from Carl
Roth GmbH&Co (Karlsruhe, Germany), sodium citrate – from Penta
(Praha, Czech Republic), hydrochloric acid 37% – from Acta Med-
ica (Hradec Kralove, Czech Republic), sodium carbonate – from
Lachema (Neratovice, Czech Republic). Before investigations glu-
cose solutions were allowed to mutarotate overnight. All other
chemicals used in the present study were either of analytical
grade or of highest quality. All solutions were prepared using
deionised water purified with water purification system Millipore
S.A. (Molsheim, France). The solution of sodium acetate buffer (SA,
0.05 mol L−1 CH3 COONa·3H2 O) with 0.1 mol L−1 KCl was prepared
by mixing sodium acetate trihydrate and potassium chloride, which
were obtained from Reanal (Budapest, Hungary) and Lachema (Ner-
atovice, Czech Republic). Pyrrole was obtained from Sigma–Aldrich
(Steinheim, Germany). Carbon (3 mm diameter, 99.999%, low den-
sity) was purchased from Sigma-Aldrich (St. Louis, USA), alumina
powder (grain size 0.3 ␮m, Type N) – from Electron Microscopy Sci-
ences (Hatfield, USA) and 25% glutaraldehyde solution – from Fluka
Chemie GmbH (Buchs, Switzerland).
2.2. Synthesis of gold nanoparticles
Gold nanoparticles of different sizes (3.5, 6 and 13 nm) were syn-
thesised by the reduction of HAuCl4 ·3H2 O by sodium citrate in the
presence of tannic acid. Aqueous solution of tetrachloroauric acid
(81 mL of 0.01% [w/v] HAuCl4 ·3H2 O) was heated in an Erlenmeyer
flask using a magnetic stirrer. Solutions of sodium citrate (4 mL of
1% [w/v]) and tannic acid in deionised water (5.0, 0.5 or 0.025 mL
of 1% [w/v] solution for 3.5, 6 or 13 nm Au-NPs respectively) were
added to the flask and heated up to 60 ◦ C stirring rapidly. After
225
preheating solutions were mixed, heated up to 98 ◦ C and kept at this
temperature for 3 min to yield sols of gold nanoparticles. The reac-
tion duration was 10 min and the mixing speed was 1000 rpm. Then
5 mL of 25 mmol L−1 Na2 CO3 was added for the neutralisation of the
solution containing 3.5 nm size Au-NPs. The size of Au-NPs (3.5, 6
and 13 nm) was measured by atomic force microscopy (AFM). The
average size of gold nanoparticles distributed on the surface of 8 Å
SiO2 substrate received from AIXTRON AG (Aachen, Germany), was
evaluated from the height-distribution histograms of AFM images.
The initial concentration of gold in all used solutions was the same
– 0.058 mg ml−1 . Stable sols of gold nanoparticles were stored in
dark glass flasks at +4 ◦ C.
2.3. Pre-treatment of the working electrode
The working surface area of carbon rod electrodes was
0.071 cm2 . Carbon rods of spectroscopic carbon were cut and pol-
ished on fine emery paper and then polished by slurry of alumina
powder containing 0.3 micron grains of Al2 O3 . After this the elec-
trode surface was rinsed with distilled water and dried at 20 ± 2 ◦ C.
Then lateral surface of the electrode was sealed in a silicone tube
to prevent contact with the solution.
2.3.1. Electrode modification by gold nanoparticles and glucose
oxidase
Four different types of electrodes were prepared: (i) modi-
fied with GOx (GOx/CR); (ii–iv) modified with Au-NPs of 3.5, 6
and 13 nm sizes and GOx (GOx/3.5Au-NPs/CR; GOx/6Au-NPs/CR;
GOx/13Au-NPs/CR, respectively).
During the preparation of the GOx/CR electrode, 3 ␮L of GOx
solution of 40 mg mL−1 concentration were deposited on the elec-
trode surface and water was evaporated by intensive ventilation at
20 ± 2 ◦ C. For the preparation of GOx/Au-NPs/CR electrodes 3 ␮L of
gold nanoparticle sol were deposited on the working electrode and
after the evaporation of water 3 ␮L of GOx solution of 40 mg mL−1
concentration were deposited additionally. After the evaporation
electrodes were kept in a closed vessel over a 25% solution of glu-
taraldehyde for 15 min at room temperature. The development and
optimisation of this immobilisation procedure was described in
detail previously [37]. Prior to all electrochemical measurements,
working electrodes were thoroughly washed with distilled water
to remove the non-cross-linked enzyme and/or gold nanoparticles.
All working electrodes were stored in a closed vessel over buffer
solution at +4 ◦ C until needed in the experiment.
2.3.2. Electrode modification by the polypyrrole layer
Chemical polymerisation of pyrrole over GOx/CR and
GOx/13Au-NPs/CR electrodes was performed in 0.05 mol L−1
SA buffer, pH 6.0, containing 0.05 mol L−1 glucose and 0.5 mol L−1
pyrrole (polymerisation solution) for a defined period (1–12 h) at
+4 ◦ C. Between measurements electrodes were stored in a closed
vessel hanging over the 0.05 mol L−1 SA, pH 6.0, at +4 ◦ C till the
next stage of polymerisation (duration of experiment 5 days).
For the evaluation of Au-NP size (3.5, 6 and 13 nm) effect on
the performance of the electrodes before and after the polypyrrole
layer formation, electrodes (GOx/3.5Au-NPs/CR; GOx/6Au-NPs/CR;
GOx/13Au-NPs/CR and GOx/CR) were immersed in the polymerisa-
tion solution for 13 h. Prior to electrochemical measurements the
electrodes were thoroughly washed with distilled water.
2.4. Electrochemical measurements
All electrochemical measurements were performed using a
computerised potentiostat PGSTAT 302 N/Autolab (EcoChemie,
Netherlands) with GPES 4.9 software in chrono amperometry
mode at +0.3 V vs. Ag/AgCl. A conventional three-electrode system
226 N. German et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 224–230

comprising working electrode (modified as described above), 2 cm2

platinum as an auxiliary electrode and Ag/AgCl with 3 mol L−1 KCl
filling solution Metrohm (Herisau, Switzerland) as a reference was
employed for all electrochemical experiments. All experiments
were performed at room temperature in SA buffer, pH 6.0, with
0.1 mol L−1 KCl while stirring (120 rpm). Electrochemical detection
of the analytical signal was performed at different concentrations
of glucose (0.1–100 mmol L−1 ) in the presence of 2 mmol L−1 of
N-methylphenazonium methyl sulphate. The activity of GOx was
estimated by measuring the re-oxidation current of the reduced
PMS form (PMSH2 ) and/or H2 O2 produced by the enzymatic
reaction at +0.3 V potential vs. Ag/AgCl. The results of all electro-
chemical measurements are reported as the mean value of three
independent experiments.

2.5. Imaging by atomic force microscopy

Tapping mode of atomic force microscopy (AFM) was used for

the imaging of differently modified surfaces. BioScope II, Veeco
Instruments Ltd. (Santa Barbara, USA) and TESP cantilevers (Veeco)
were used for all AFM experiments. Experimental data were pro-
cessed by “diNanoScope 7.30” and “Gwyddion 2.10 NT-MDT Nova”
programs.

2.6. Calculations

Amperometric signals showed hyperbolic dependence on glu-

cose concentration, this was in agreement with Michaelis–Menten
kinetics. The kinetic parameters, i.e., the maximal current gener-
ated during enzymatic reaction (Imax ) and the apparent Michaelis
constant (KM(apparent) ) are represented by a and b parameters of
the hyperbolic function y = ax/(b + x) used for the approximation of
results. The kinetic parameters of the enzyme catalysed reaction
were calculated using SigmaPlot software.

3. Results and discussion

The Au-NPs were synthesised according to the protocol pre-

sented in experimental part and dry samples were investigated
by AFM using tapping mode. AFM images illustrate the size-
distribution of Au-NPs of different sizes (Fig. 1). Shape of height
histogram of Au-NPs shows that formed Au-NPs is nearly monodis-
persed since distribution in diameter of 13, 6 and 13 nm Au-NPs is
narrow, within the range of 12–16 nm, 5–7 nm and 2–5 nm, respec-
tively (Fig. 1).
Four types of amperometric biosensors based on carbon rod
electrodes were designed and compared during the chemical poly-
merisation of pyrrole. One type of working electrode was a CR
electrode with immobilised GOx, other three types of electrodes
were CR electrodes modified with Au-NPs of different sizes (3.5,
6 or 13 nm) and GOx [38]. Amperometric measurements clearly
demonstrated that the biological activity of GOx was retained dur-
ing the immobilisation procedure; it is in line with the results of
other investigations where the same immobilisation procedure was
used [38].
During enzymatic reaction electrons are transferred towards the
positively charged electrode and steady-state currents are regis-
tered. The reactions and electron transfer routes, which take place
because of the catalytic action of GOx/Au-NPs/CR electrodes, are
presented in Fig. 2a. PMS serves as a redox mediator and re-oxidases
the active site of GOx. In this case electron transfer via a reduced
form of mediator (PMSH2 ) may occur in two ways: directly to the
CR electrode (I way) or in combination of PMSH2 with Au-NPs (II
way) as shown in other studies [4,5,39]. The major advantage of
GOx/Au-NPs/CR electrode is advanced electrochemical activity due
to Au-NPs compared with that of a non-modified carbon electrode.
Fig. 1. Size distribution histograms of 13 (a), 6 (b) and 3.5 nm (c) Au-NPs.
This results the increase of electrocatalytic current [2], therefore
the sensitivity of amperometric biosensors becomes higher [38].
According to previous results [40] GOx/13Au-NP/CR electrode gives
1.8 times higher amperometric signal when compared with GOx/CR
electrode [38].
GOx immobilised on the electrode in the presence of glucose
and dissolved oxygen generated hydrogen peroxide and glucono-
lactone, which was hydrolysed to gluconic acid [38]. During this
reaction the pH locally decreased close to the GOx active site while
hydrogen peroxide concentration locally increased. Therefore low
pH value and high concentration of oxidator near the enzyme
created optimal conditions for the polymerisation of polypyrrole.
During the enzymatic polymerisation GOx is covered by polymer
(Fig. 2b), negatively charged GOx at pH 6.0 and positively charged
polymer chains electrostatic interaction takes place [17]. Strong
interaction between Ppy chains enables aggregation of newly com-
posed chains with previously formed ones [41]. After the formation
 N. German et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 224–230
Fig. 2. Schematic representation of polypyrrole-coated gold nanoparticles and glucose oxidase in PMS mediated biosensor design.
of Ppy layer the influence of this layer on the sensitivity of modi-
fied electrodes was investigated. In this case III-rd additional way
of electron transfer via ␲–␲ conjugated polymer becomes possible
(Fig. 2b).
The polymerisation duration had a significant influence on the
sensitivity of electrodes. Hyperbolic dependences of amperomet-
ric signals on the concentration of glucose in the range from
0.1 to 100 mmol L−1 were observed at Ppy/GOx/CR (Fig. 3) and
Ppy/GOx/13Au-NPs/CR (Fig. 4) electrodes at different stages of Ppy
layer formation.
To form Ppy layer electrodes were kept in polymerisation solu-
tion for 1, 3, 7 and 12 h. The maximal current generated by the
enzymatic reaction during different stages of polymer layer for-
mation and the apparent Michaelis constant are presented in
Figs. 3b, c and 4b, c for Ppy/GOx/CR and Ppy/GOx/13Au-NPs/CR
electrodes respectively.
During the chemical polymerisation both electrodes were cov-
ered by a layer of polypyrrole. After the 12-h polymerisation Imax
decreased by 18.5 and 8.9 times while KM(apparent) increased by
7.75 and 7.37 times for Ppy/GOx/CR and Ppy/GOx/Au-NPs/CR elec-
trodes, respectively. The increment of KM(apparent) by more than 7
times after polymerisation might be considered as an evidence that
GOx immobilised on the carbon rod electrode surface was wrapped
within the formed polypyrrole layer. However, these results illus-
trate that gold nanoparticles have only minor influence on the
Michaelis constant after polymer layer formation.
a b
1 50
50
50
Imax, μA
40 2 25
30
ΔI, μA
0 3
c
-1
20 3 75
KM, mmol L
50
10
4 25
5
0 0
0 25 50 75 100 0 4 8 12
C(Glu), mmol L-1 Time, hours
Fig. 3. Calibration plots of Ppy/GOx/CR electrode registered at different stages of
polypyrrole layer formation (a); changes of maximal current (b) and the apparent
Michaelis constant (c) (1 curve – amperometric signals registered before the start of
polymerisation; 2–5 curves – after 1, 3, 7 and 12 h of polymerisation, respectively,
in 0.05 mol L−1 SA, pH 6.0, with 0.05 mol L−1 glucose and 0.5 mol L−1 pyrrole).
227
The duration of polymerisation process influences the linear
detection range of Ppy modified electrodes, e.g., it was deter-
mined that Ppy/GOx/CR and Ppy/GOx/13Au-NPs/CR electrode
linear ranges of detection increased along with extended poly-
merisation duration. The linear response range of Ppy/GOx/CR
and Ppy/GOx/13Au-NPs/CR electrodes modified by 3 h Ppy for-
mation can be extended to not less than 20 mmol L−1 of glucose,
which is at least twice higher than that of unmodified elec-
trodes (10 mmol L−1 ). The extension of linear range towards higher
concentrations of glucose usually is related to decrease of sen-
sitivity. Estimated Imax (Figs. 3b and 4b) and KM(apparent) values
(Figs. 3c and 4c) illustrate that the increase of KM(apparent) up to
50 mmol L−1 for Ppy/GOx/CR electrode was followed by decrease
of sensitivity for 7.8 times, while for Ppy/GOx/13Au-NPs/CR elec-
trode the same increase of KM(apparent) was followed by decrease of
sensitivity for 6.2 times. Such significant increase of KM(apparent) for
both Ppy/GOx/CR and Ppy/GOx/Au-NPs/CR electrodes is followed
by increase of linear analyte detection intervals and such modified
electrodes might be applied for the detection of glucose concentra-
tion in real samples where glucose concentration is in the range of
1.5–20 mmol L−1 .
The limit of detection of glucose was determined for both types
of electrodes and was equal to 0.07 mmol L−1 at a signal to noise
ratio of 3 standard deviations. Reproducibility of Ppy/GOx/CR and
a 1 b 75
60
Imax, μA
2 25
40
ΔI, μA
0
c
75
KM, mmol L-1
20
50
4 25
5
0 0
0 25 50 75 100 0 4 8 12
C(Glu), mmol L-1 Time, hours
Fig. 4. Calibration plots of GOx/13Au-NPs/CR electrode registered at different stages
of polypyrrole layer formation (a); changes of maximal current (b) and the appar-
ent Michaelis constant (c) (1 curve – amperometric signals registered before the
initiation of polymerisation; 2–5 curves – after 1, 3, 7 and 12 h of polymerisation,
respectively, in 0.05 mol L−1 SA, pH 6.0, with 0.05 mol L−1 glucose and 0.5 mol L−1
pyrrole).
228 N. German et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 224–230

Fig. 5. AFM images (a and c) and height-distribution diagrams (b and d) of GOx/13Au-NPs/CR electrode unmodified (a and b) and modified (c and d) with Ppy after a 12 h
polymerisation in 0.05 mol L−1 SA, pH 6.0, with 0.05 mol L−1 glucose and 0.5 mol L−1 pyrrole.

Ppy/GOx/13Au-NPs/CR electrodes at larger values of glucose con-
centration (20 mmol L−1 ) was 12.5 and 9.4% respectively. Better
reproducibility (4.2%) using 2.6 nm Au-NPs and Ppy demonstrated
by other authors may be explained by proper orientation of GOx
molecules, which are more firmly adsorbed on the surface of Au-
NPs [42].
Variations in height of 16.5 ± 6 nm appear on GOx/13Au-NPs/CR
electrode surface and these features can be explained as clusters of
Au-NPs and GOx. It was found that gold nanoparticles penetrated
into the micro-gaps of carbon rod electrode. 13 nm Au-NPs cov-
ered the surface of carbon forming continuous layer and for this
reason the electrode surface became smoother. Additional modifi-
cation of the same surface with enzyme showed that the height of
most nano-features slightly increased to ∼16.5 nm (Fig. 5a and b).
AFM investigations showed that after 12 h of chemical polymeri-
sation the surface of GOx/13Au-NPs/CR electrode was covered by
polypyrrole, which was unequally distributed over the electrode.
Ppy-based features of about 700 nm in height appeared after the
12 h polymerisation period (Fig. 5c and d).
In previous work [38] it was estimated that the sensitivity
of established electrochemical systems depends on the size of
Au-NPs immobilised on the surface of the carbon rod electrode:
GOx/Au-NPs/CR electrode with 3.5 and 6 nm of Au-NPs showed
higher electrocatalytic activity in comparison with 13 nm Au-NPs.
This effect was proved in present research by the calculation of
KM(apparent) and Imax . In systems based on 3.5, 6 and 13 nm Au-NPs
Imax was 2.14, 2.07 and 1.86 times higher in comparison with the
GOx based system. The sublayer based on smaller Au-NPs has a
larger effective surface area, and the transfer of electrons between
GOx and electrode becomes more efficient compared with that
observed for electrodes based on larger Au-NPs or not modified
by Au-NPs. Insignificant differences of KM(apparent) were observed
with decreasing size of Au-NPs (from 13 to 3.5 nm).
Nonetheless, it was investigated that the size of Au-NPs has
a significant influence on Michaelis–Menten kinetics after the
same duration of Ppy layer formation (Table 1). After 13 h of
enzymatic polymerisation Imax decreased by 1.66, 2.07, 2.14 and
1.53 times for the Ppy/GOx/3.5Au-NPs/CR, Ppy/GOx/6Au-NPs/CR,
Ppy/GOx/13Au-NPs/CR and Ppy/GOx/CR electrodes, respectively, if
compared with electrodes not covered by Ppy. The decrease of Imax
depends on the size of Au-NPs: smaller Au-NPs (3.5 nm) have less
effect if compared with larger Au-NPs (13 nm). This effect might
be explained by increased electrochemically effective area of elec-
trode, which is modified with Au-NPs, and surface-area-dependent
facilitation of electron transfer efficiency. Electrodes modified with
smaller Au-NPs showed higher amperometric responses before and
after the polymer layer formation if compared with electrodes
modified with larger Au-NPs. Nevertheless, the analytical signal of
electrodes modified with Au-NPs of different sizes before and after
Ppy formation is higher if compared with electrode Ppy/GOx/CR,
 N. German et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 224–230
Table 1
KM(apparent) and Imax calculated for GOx/Au-NPs/CR electrodes based on adsorbed 3.5, 6, 13 nm Au-NPs and for GOx/CR electrode (glucose concentration ranged from 0.1 to
100 mmol L−1 ; polymerisation solution – 0.05 mol L−1 SA, pH 6.0, with 0.05 mol L−1 glucose and 0.5 mol L−1 pyrrole).
Immobilisation and modification of CR electrode Duration of Ppy formation, h
0 76.2
GOx/3.5Au-NPs/CR electrode
13
0 73.8
GOx/6Au-NPs/CR electrode
13
0 66.2
GOx/13Au-NPs/CR electrode
13
0 35.6
GOx/CR electrode
13
which was designed without Au-NPs. The presence of Au-NPs influ-
ences the value of KM(apparent) after the chemical polymerisation of
Ppy. After 13 h of enzymatic polymerisation performed in order to
get Ppy/GOx/3.5Au-NPs/CR, Ppy/GOx/6Au-NPs/CR, Ppy/GOx/13Au-
NPs/CR and Ppy/GOx/CR electrodes the KM(apparent) registered by
these electrodes increased by 2.22, 2.26, 2.36 and 1.74 times,
respectively. Results illustrate that the most significant increase of
KM(apparent) was registered if Au-NPs-modified electrode was cov-
ered by polypyrrole.
4. Conclusions
Au-NPs in a combination with enzymatically formed polypyr-
role layer offered some advantages for the design of electrochemi-
cal biosensors. The limit of detection for glucose using Ppy/GOx/CR
and Ppy/GOx/13Au-NPs/CR electrodes after 3 h of chemical poly-
merisation was determined to be 0.07 mmol L−1 . The sensitivity
of analytical systems modified with polypyrrole depended on the
sizes of Au-NPs. Smaller Au-NPs based systems exhibited higher
amperometric responses at the same concentration of glucose
before and after the formation of polypyrrole layer.
Acknowledgement
This work was financially supported by Lithuanian State Sci-
ence and Studies Foundation project number S-6/2007 and COST
program D43.
References
[1] J. Wang, Nanomaterial-based electrochemical biosensors, Analyst 130 (2005)
421–426.
[2] F. Wang, S. Hu, Electrochemical sensors based on metal and semiconductor
nanoparticles, Microchim. Acta 165 (2009) 1–22.
[3] A.C. Barton, S.D. Collyer, F. Davis, D.D. Gornall, K.A. Law, E.C.D. Lawrence, D.W.
Mills, S. Myler, J.A. Pritchard, M. Thompson, S.P.J. Higson, Sonochemically fab-
ricated microelectrode arrays for biosensors offering widespread applicability:
Part I, Biosens. Bioelectron. 20 (2004) 328–337.
[4] N. German, A. Ramanavicius, J. Voronovic, Y. Oztekin, A. Ramanaviciene, The
effect of colloidal solutions of gold nanoparticles on the performance of a glu-
cose oxidase modified carbon electrode, Microchim. Acta 172 (2011) 185–191.
[5] M. Pumera, S. Sánchez, I. Ichinose, J. Tang, Electrochemical nanobiosensors,
Sens. Actuators B 123 (2007) 1195–1205.
[6] Y. Wang, H. Xu, J. Zhang, G. Li, Electrochemical sensors for clinical analysis,
Sensors 8 (2008) 2043–2081.
[7] A. Ramanavicius, A. Ramanaviciene, A. Malinauskas, Electrochemical sensors
based on conducting polymer – polypyrrole, Electrochim. Acta 51 (2006)
6025–6037 (Review).
[8] Md.A. Rahman, P. Kumar, D.-S. Park, Y.-B. Shim, Electrochemical sensors based
on organic conjugated polymers, Sensors 8 (2008) 118–141 (Review).
[9] A. Ramanaviciene, A. Ramanavicius, Application of polypyrrole for the creation
of immunosensors, Crit. Rev. Anal. Chem. 32 (2002) 245–252.
[10] M. Singh, P.K. Kathuroju, N. Jampana, Polypyrrole based amperometric glucose
biosensors, Sens. Actuators B 143 (2009) 430–443.
[11] W. Shi, Y. Sahoo, M.T. Swihart, Gold nanoparticles surface-terminated with
bifunctional ligands, Colloids Surf. A: Physicochem. Eng. Asp. 246 (2004)
109–113.
[12] P. Yáñez-Sedeño, J.M. Pingarrón, Gold nanoparticles-based electrochemical
biosensors, Anal. Bioanal. Chem. 382 (2005) 884–886.
229
Imax , ␮A KM(apparent) , mmol L−1 R2
19.7 0.9877
46.0 43.7 0.9921
21.3 0.9882
35.7 48.1 0.9920
19.9 0.9862
30.9 46.9 0.9922
17.4 0.9751
23.3 30.3 0.9768
[13] X. Luo, A. Morrin, A.J. Killard, M.R. Smyth, Application of nanoparti-
cles in electrochemical sensors and biosensors, Electroanalysis 18 (2006)
319–326.
[14] I. Willner, B. Willner, E. Katz, Biomolecule-nanoparticle hybrid systems for
bioelectronic applications, Bioelectrochemistry 70 (2007) 2–11.
[15] E. Bakker, Y. Qin, Electrochemical sensors, Anal. Chem. 78 (2006)
3965–3983.
[16] F. Kurniawan, V. Tsakova, V.M. Mirsky, Gold nanoparticles in nonenzymatic
electrochemical detection of sugars, Electroanalysis 18 (2006) 1937–1942.
[17] X. Cui, J. Wiler, M. Dzaman, R.A. Altschuler, D.C. Martin, In vivo stud-
ies of polypyrrole/peptide coated neutral probes, Biomaterials 24 (2003)
777–787.
[18] A. Ramanaviciene, A. Ramanavicius, Towards the hybrid biosensors based
on biocompatible conducting polymers, in: M.S. Shur, A. Žukauskas (Eds.),
UV Solid-State Light Emitters and Detectors, Kluwer Academic Publishers,
Netherlands, 2004, pp. 287–296.
[19] A. Ramanaviciene, A. Ramanavicius, Affinity sensors based on nano-structured
␲–␲ conjugated polymer polypyrrole, in: D.W. Thomas (Ed.), Advanced Bio-
materials for Medical Applications, Kluwer Academic Publishers, Netherlands,
2004, pp. 111–125.
[20] A. Ramanaviciene, A. Ramanavicius, Molecularly imprinted polypyrrole-based
synthetic receptor for direct detection of bovine leukemia virus glycoproteins,
Biosens. Bioelectron. 20 (2004) 1076–1082.
[21] V. Rumbau, R. Marcilla, E. Ochoteco, J.A. Pomposo, D. Mecerreyes, Ionic liq-
uid immobilized enzyme for biocatalytic synthesis of conducting polyaniline,
Macromolecules 39 (2006) 8547–8549.
[22] E. Granot, E. Katz, B. Basnar, I. Willner, Enhanced bioelectrocatalysis using Au-
nanoparticle/polyaniline hybrid systems in thin films and microstructured rods
assembled on electrodes, Chem. Mater. 17 (2005) 4600–4609.
[23] A. Chyla, D.J. Walton, C. Hall, Electrochemical oxidation and reduction on
polypyrrole electrodes, Synth. Met. 37 (1990) 115–122.
[24] R.N. Singh, M. Malviya, P. Chartier, Electrochemical characterization of com-
posite films of LaNiO3 and polypyrrole for electrocatalysis of O2 reduction, J. N.
Mater. Electrochem. Syst. 10 (2007) 181–186.
[25] J. Njagi, S. Andreescu, Stable enzyme biosensors based on chemically syn-
thesized Au-polypyrrole nanocompsites, Biosens. Bioelectron. 23 (2007)
168–175.
[26] Yu. Xian, Y. Hu, F. Liu, Y. Xian, H. Wang, L. Jin, Glucose biosensor based on Au
nanoparticles-conductive polyaniline nanocomposite, Biosens. Bioelectron. 21
(2006) 1996–2000.
[27] R. Cruz-Silva, L. Arizmendi, M. Del-Angel, J. Romero-Garcia, pH- and thermosen-
sitive polyaniline colloidal particles prepared by enzymatic polymerisation,
Langmuir 23 (2007) 8–12.
[28] A.F. Diaz, B. Hall, Mechanical properties of electrochemically prepared polypyr-
role films, IBM J. Res. Dev. 27 (1983) 342–347.
[29] A. Ramanavicius, A. Kausaite, A. Ramanaviciene, J. Acaite, A. Malinauskas, Redox
enzyme – glucose oxidase – initiated synthesis of polypyrrole, Synth. Met. 156
(2006) 409–413.
[30] A. Ramanavicius, A. Kausaite, A. Ramanaviciene, Self-encapsulation of oxidases
as a basic approach to tune upper detection limit of amperometric biosensors,
Analyst 133 (2008) 1083–1089.
[31] A. Kausaite-Minkstimiene, V. Mazeiko, A. Ramanaviciene, A. Ramanavicius,
Evaluation of amperometric glucose biosensors based on glucose oxidase
encapsulated within enzymatically synthesized polyaniline and polypyrrole,
Sens. Actuators B: Chem. 158 (2011) 278–285.
[32] M.L. Mena, P. Yáñez-Sedeño, J.M. Pingarrón, A comparison of different strate-
gies for the construction of amperometric enzyme biosensors using gold
nanoparticle-modified electrodes, Anal. Biochem. 336 (2005) 20–27.
[33] A. Kausaite-Minkstimiene, V. Mazeiko, A. Ramanaviciene, A. Ramanavicius,
Enzymatically synthesized polyaniline layer for extension of linear detection
region of amperometric glucose biosensor, Biosens. Bioelectron. 26 (2010)
790–797.
[34] A. Ramanaviciene, A. Ramanavicius, Pulsed amperometric detection of DNA
with an ssDNA/polypyrrole-modified electrode, Anal. Bioanal. Chem. 379
(2004) 287–293.
[35] A. Ramanaviciene, A. Kausaite, S. Tautkus, A. Ramanavicius, Biocompatibility of
polypyrrole particles: an in vivo study in mice, J. Pharm. Pharmacol. 59 (2007)
311–315.
[36] D. Wei, A. Ivaska, Electrochemical biosensors based on polyaniline, Chem. Anal.
(Warsaw) 51 (2006) 839–852.
230 N. German et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 224–230

[37] A. Ramanavicius, Amperometric biosensor for the determination of creatine,
Anal. Bioanal. Chem. 387 (2007) 1899–1906.
[38] N. German, A. Ramanaviciene, J. Voronovic, A. Ramanavicius, Glucose biosensor
based on graphite electrodes modified with glucose oxidase and colloidal gold
nanoparticles, Microchim. Acta 168 (2010) 221–229.
[39] Y. Xiao, F. Patolsky, E. Katz, J.F. Hainfeld, I. Willner, Plugging into enzymes:
nanowiring of redox enzymes by a gold nanoparticle, Science 299 (2003)
1877–1881.
[40] B. Neiman, E. Grushka, O. Lev, Use of gold nanoparticles to enhance capillary
electrophoresis, Anal. Chem. 73 (2001) 5220–5227.
[41] E.J. Oh, K.S. Jang, A.G. MacDiarmid, High molecular weight soluble polypyrrole,
Synth. Met. 125 (2002) 267–272.
[42] S. Zhang, N. Wang, H. Yu, Y. Niu, C. Sun, Covalent attachment of glucose oxi-
dase to an Au electrode modified with gold nanoparticles for use as glucose
biosensor, Bioelectrochemistry 67 (2005) 15–22.