Anal. Chem.
2008, 80, 8253–8259
Following the Biocatalytic Activities of Glucose
Oxidase by Electrochemically Cross-Linked
Enzyme-Pt Nanoparticles Composite Electrodes
Lily Bahshi,† Marco Frasconi,‡ Ran Tel-Vered,† Omer Yehezkeli,† and Itamar Willner*,†
Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, and Dipartimento di Studi di
Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita “La Sapienza”, Rome, Italy
An integrated platinum nanoparticles (NPs)/glucose oxi-
dase (GOx) composite film associated with a Au electrode
is used to follow the biocatalytic activities of the enzyme.
The film is assembled on a Au electrode by the electropo-
lymerization of thioaniline-functionalized Pt NPs and
thioaniline-modified GOx. The resulting enzyme/Pt NPs-
functionalized electrode stimulates the O2 oxidation of
glucose to gluconic acid and H2O2. The modified electrode
is then implemented to follow the activity of the enzyme
by the electrochemical monitoring of the generated H2O2.
The effect of the composition of the Pt NPs/GOx cross-
linked nanostructures and the optimal conditions for the
preparation of the electrodes are discussed.
Numerous oxidases catalyze the oxidation of their specific
substrates by molecular oxygen with the concomitant generation
of H2O2 (e.g., glucose oxidase, lactate oxidase, choline oxidase,
or cholesterol oxidase). Accordingly, numerous electrochemical,1
optical,2-4 or chemiluminescent5,6 biosensors for the different
substrates were developed by probing the H2O2 generated by the
respective biotransformations. Specifically, the electrocatalytic
reduction of H2O2 by horseradish peroxidase-functionalized
electrodes7-9 or other hemoprotein-modified electrodes10 were
used to develop biosensors for H2O2 and for the substrates of
different oxidases. Similarly, different metals11,12 or transition
* To whom correspondence should be addressed. E-mail: willnea@vms.huji.ac.il.
Phone: +972-2-6585272. Fax: +972-2-6527715.
†
The Hebrew University of Jerusalem.
‡
Universita “La Sapienza”.
(1) Wang, J. Chem. Rev. 2008, 108, 814–825.
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47, 1676–1679.
(5) Shi, C. G.; Xu, J. J.; Chen, H. Y. J. Electroanal. Chem. 2007, 610, 186–192.
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Electrochem. Commun. 2000, 2, 441–444.
10.1021/ac801398m CCC: $40.75  2008 American Chemical Society
Published on Web 10/08/2008
metal complexes13-16 acted as catalysts for the electrocatalytic
reduction of H2O2 and for the development of biosensors for
different substrates of oxidases. Furthermore, the development
of glucose-sensing electrodes employing glucose oxidase as a
biocatalyst attracted extensive research efforts. Different enzyme
electrodes that monitored the biocatalytically generated H2O2 were
used to electrochemically analyze glucose.17 Similarly, electrically
contacted glucose oxidase electrodes prepared by the tethering
of a redox relay to the protein18 or the incorporation of glucose
oxidase in redox-active conductive polymers associated with
electrodes19 were implemented for glucose sensing. Nanobiotech-
nology has introduced new tools for biosensor technologies, and
biomolecule-nanoparticle (NP) hybrid systems were widely
applied to develop biosensors.20 Au NPs conjugated to redox
enzymes were used to electrically contact the redox sites of the
biocatalysts with electrodes and to activate their bioelectrocatalytic
functions.21,22 The catalytic enlargement of Au NPs associated with
electrodes enhanced the conductivity at the enzyme-modified
electrode surfaces, and this improved the bioelectrocatalytic
functions of the modified electrodes.23 Similarly, different
biomolecule-metal NP hybrids were used as catalytic labels for
the amplified detection of biorecognition events through the
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Acta 1982, 142, 277–279. (c) Urban, G.; Jobst, G.; Kohl, F.; Jachimowicz,
A.; Olcaytug, F.; Tilado, O.; Goiser, P.; Nauer, G.; Pittner, F.; Schalkhammer,
T.; Mann-Buxbaum, E. Biosens. Bioelectron. 1991, 6, 555–562. (d) Laury,
J. P.; McAteer, K.; Atrash, S. E.; O’Neill, R. D. J. Chem. Soc., Chem. Commun.
1994, 2483–2484. (e) Alfonta, L.; Katz, E.; Willner, I. Anal. Chem. 2000,
72, 927–935.
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A. J. Chem. Soc. Faraday Trans. 1996, 20, 4131–4136.
(20) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042–6108.
(21) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. Science 2003,
299, 1877–1881.
(22) Zayats, M.; Katz, E.; Baron, R.; Willner, I. J. Am. Chem. Soc. 2005, 127,
12400–12406.
(23) Yan, Y.; Tel-Vered, R.; Yehezkeli, O.; Cheglakov, Z.; Willner, I. Adv. Mater.
2008, 20, 2365–2370.
Analytical Chemistry, Vol. 80, No. 21, November 1, 2008 8253
Scheme 1. (A) Preparation of a Pt NPs Monolayer Assembly through Covalent Binding of
Thioaniline-Modified Pt NPs to a Mercaptopropionic Acid-Modified Au Electrode. (B) Synthesis of a 3D
Oligoaniline-Cross-Linked Pt NPs Structure by the Electropolymerization of the Thioaniline-Modified Pt
Nanoparticles on the Thioaniline-Modified Au Electrodes

electrocatalyzed reduction of H2O224 or the H2O2-mediated genera-
tion of chemiluminescence.25 Also, enzyme-carbon nanotube
hybrid systems were used to develop glucose-sensing electrodes.26
Alternatively, the catalytic growth of Au NPs by enzyme-generated
H2O2 enabled the optical, colorimetric, determination of enzyme
activities,27 their substrates,28 or their inhibitors.29 Here we report
on a novel method to assemble composite Pt NPs and glucose
oxidase (GOx) films on Au electrodes by the electropolymerization
of thioaniline-functionalized Pt NPs and thioaniline-modified GOx.
The resulting Pt NPs/GOx arrays are cross-linked by oligoaniline
bridges during the electropolymerization process. The modified
electrode reveals several unique functions that advance biosensor
technology: (i) The Pt particles reveal high electrocatalytic activity
toward the electrocatalytic reduction of H2O2 generated by the
GOx-mediated oxidation of glucose. (ii) The preparation of the
functionalized electrode by electropolymerization allows the ad-
dressing of a selective working electrode in a miniaturized three-
electrode configuration and the high-throughput preparation of
numerous miniaturized cells. (iii) The electropolymerization
process enables control over the sensitivity of the resulting bio-
electrocatalytic matrixes by regulating the content of the Pt NPs/
enzyme composite.
Pt NPs were capped with a mixed monolayer of thioaniline
and mercaptoethanesulfonic acid. While the thioaniline provides
the electropolymerizable monomer units, the mercaptoethane-
sulfonate units enhance the stability of the Pt NPs against
aggregation and precipitation in aqueous media. In a primary
experiment, the functionalized Pt NPs were covalently tethered
to Au electrodes that were functionalized with mercaptopropionic
acid, Scheme 1A, to form a two-dimensional monolayer of Pt NPs.
Figure 1 shows the cyclic voltammograms of the Pt NPs-modified
electrode upon addition of H2O2. Evidently, in the presence of
H2O2, electrocatalytic cathodic waves are observed (the overpo-
tential for the reduction of H2O2 at the Au electrode is reduced),
and as the concentration of H2O2 is elevated, the intensities of
the electrocatalytic currents are enhanced. In a control experi-
ment, no electrocatalytic currents could be observed at a bare
Au electrode lacking the Pt NPs within this potential range.
These results suggest that the Pt NPs, indeed, electrocatalyze the
reduction of H2O2. Figure 1, inset, depicts the calibration curve
corresponding to the amperometric response of the Pt NPs-
modified electrode at E ) -0.35 V versus saturated calomel
electrode (SCE), in the presence of variable concentrations of
H2O2.
In the subsequent experiment, the thioaniline-modified Pt NPs
were electropolymerized on thioaniline-functionalized Au elec-
trodes, Scheme 1B. The electropolymerization of a thioaniline
monolayer associated with a Au surface was reported previously,30
and the generation of a two-dimensional conductive polymer on
the surface was demonstrated. Also, a thioaniline-modified mono-
layer associated with Au electrode, in a densely packed configu-
ration31 or in a diluted state,32 enabled the electrochemical

(24) Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Anal. Chem. 2006, 78, 2268–
2271.
(25) Gill, R.; Polsky, R. I. Willner, Small 2006, 2, 1037–1041.
(26) (a) Wang, J.; Musameh, M.; Lin, Y. J. Am. Chem. Soc. 2003, 125, 2408–
2409. (b) Wang, J.; Musameh, M. Analyst 2003, 128, 1382–1385. (c) Lin,
Y.; Lu, F.; Tu, Y.; Ren, Z. Nano Lett. 2004, 2, 191–195. (d) Patolsky, F.;
Weizmann, Y.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 2113–2117. (e)
Yan, Y.; Yehezkeli, O.; Willner, I. Chem. Eur. J. 2007, 13, 10168–10175.
(27) Baron, R.; Zayats, M.; Willner, I. Anal. Chem. 2005, 77, 1566–1571.
(28) Zayats, M.; Baron, R.; Popov, I.; Willner, I. Nano Lett. 2005, 5, 21–25.
(29) Parlov, V.; Xiao, Y.; Willner, I. Nano Lett. 2005, 5, 649–653.
Figure 1. Cyclic voltammograms corresponding to the Pt NPs
monolayer-modified Au electrode in the presence of different con-
centrations of hydrogen peroxide: (a) 0.0, (b) 0.4, (c) 0.8, (d) 1.2, (e)
2.0, (f) 3.0, (g) 4.0, (h) 5.0, (i) 6.0, and (j) 7.0 mM. Scan rate 10
mV · s-1. Inset: calibration curve corresponding to the electrocatalytic
currents measured at E ) -0.35 V, using variable concentrations of
hydrogen peroxide. All measurements were performed in 0.1 M
phosphate buffer solution, pH 7.4.

8254 Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

deposition of aniline on the surface. These studies suggested that
the thioaniline-functionalized monolayer associated with the
electrode could activate the electrochemical deposition of the
thioaniline-modified particles. In the present electropolymerization
process, the Pt NPs provide the conductivity for the three-
dimensional polymerization and cross-linking of the particles. The
Pt NPs are modified with thioaniline/mercaptoethanesulfonic acid
at a ratio of 1:4. The coverage of thiolates on a 2-nm Pt NP was
estimated to be ∼172 thiolates per particle,33 and assuming that
the composition of the adsorbed mixed monolayer is similar to
the molar ratio of the thiolates in solution, we estimate that ∼34
thioaniline units are associated with each Pt NP. The electropo-
lymerization of the thioaniline-functionalized NPs yields redox-
active bridging units characteristic to conductive oligoaniline
chains (see Supporting Information, Figure S1). Although we do
not know the precise structure of the electroactive bridging units
between the Pt NPs, the dilute coverage of the NPs with the
electropolymerizable thioaniline units suggests that bisaniline
bridges are formed, although higher oligoaniline bridges cannot
be excluded. Also, microgravimetric quartz crystal microbalance
measurements indicated that the deposition of the Pt NPs on a
thioaniline-modified Au/quartz crystal resulted in a mass change
corresponding to 9.8 × 10-7 g · cm-2. Assuming a bisaniline bridge
structure, and a coverage of 34 thioaniline units per particle, the
microgravimetric analysis suggests a particle coverage of 3.8 ×
1013 particles · cm-2. Using the dimensions of a single Pt NP as
2.7 nm (Pt NPs core plus thiolated shell), this value translates to
∼3.5 random densely packed monolayers of Pt NPs that constitute
the bridged aggregated structure of the film.
Figure 2A shows the cyclic voltammograms observed upon
Figure 2. (A) Cyclic voltammograms corresponding to the oligoa-
analyzing different concentrations of H2O2 by the Pt NPs-cross- niline-cross-linked Pt NPs-functionalized Au electrode in the presence
linked electrode, produced by 60 electropolymerization cycles. of variable concentrations of hydrogen peroxide: (a) 0.0, (b) 0.05, (c)
Figure 2A, inset, depicts the derived calibration curve, where the 0.1, (d) 0.2, (e) 0.4, (f) 0.8, (g) 1.2, (h) 2.0, (i) 3.0, j) 4.0, k) 5.0, and
amperometric responses at E ) -0.35 V versus SCE are plotted (l) 6.0 mM. Scan rate 10 mV · s-1. The electrodes were prepared by
the application of 60 cyclic voltammetry scans between -0.1 and 1.1
as a function of the H2O2 concentration in the system. The
V versus SCE at 100 mV · s-1. Inset: calibration curve corresponding
effectiveness of the 3D Pt NP-functionalized electrode toward the to the electrocatalytic currents measured at E ) -0.35 V at variable
analysis of H2O2 is shown in Figure 2B. The amperometric concentrations of hydrogen peroxide. (B) Comparison of the catalytic
responses of the cross-linked Pt NPs composite are up to 30-fold currents obtained at variable concentrations of hydrogen peroxide
higher than the current responses generated by the Pt NPs using (a) the oligoaniline-cross-linked Pt NPs-modified Au electrode
and (b) the Pt NPs monolayer-modified Au electrode. All measure-
monolayer-functionalized electrode. The higher currents observed
ments were performed in a 0.1 M phosphate buffer solution, pH 7.4.
with the Pt NPs composite electrode, as compared to the Pt NPs
monolayer-functionalized electrode, may be attributed to the
higher content of the Pt NPs in the 3D electropolymerized concentrations of glucose and GOx as biocatalyst, in a homoge-
composite. neous aqueous phase (see Supporting Information, Figure S2).
The success to enhance the sensitivity of analysis toward H2O2 For any practical utility, the enzyme and the electrocatalytic
by the Pt NPs-modified electrodes suggested that the electrode Pt NPs should be integrated with the electrode. Toward this goal,
could be applied for the analysis of glucose in the presence of GOx was functionalized with electropolymerizable thioaniline units
glucose oxidase. As GOx oxidizes glucose by O2 to form gluconic according to Scheme 2A. The primary functionalization of the
acid and H2O2, the generated H2O2 relates to the concentration lysine residues with the bridging maleimide-active ester, 1, units,
of glucose, and its electrochemical determination by the electro- was followed by the Michael addition of thioaniline to the
catalytic electrode provides a quantitative measure for glucose. maleimide residues. The average loading of GOx with thioaniline
Indeed, we find that the electrode modified with the Pt NPs was estimated to be 7-8 molecules per enzyme. The activity of
composite can sense H2O2 generated in the presence of different the resulting thioaniline-functionalized GOx indicated ∼95% of the
activity of the native GOx, prior to the modification. The integrated
(30) Qing, J. X.; Xiao, L. G.; You, Y. Z.; Man, C. X.; Ming, M. Chin. Chem. Lett. Pt NPs-GOx composite electrode was prepared by the electropo-
1999, 10, 63–66. lymerization of the thioaniline-modified Pt NPs in the presence
(31) Niu, L.; Kvarnström, C.; Ivaska, A. J. Electroanal. Chem. 2007, 600, 95– of the thioaniline-functionalized GOx, Scheme 2B. The electropo-
102.
(32) Abaci, S.; Shannon, C. Electrochim. Acta 2005, 50, 2967–2973. lymerized Pt NPs provide the 3D conductivity for the covalent
(33) Eklund, S. E.; Cliffel, D. E. Langmuir 2004, 20, 6012–6018. electrochemical attachment of the GOx units. It should be noted
Analytical Chemistry, Vol. 80, No. 21, November 1, 2008 8255
Scheme 2. (A) Modification of Glucose Oxidase with an Electropolymerizable Thioaniline Function. (B)
Synthesis of a 3D Oligoaniline-Cross-Linked GOx/Pt NPs Composite by the Electropolymerization of
the Thioaniline-Modified GOx and Thioaniline-Modified Pt Nanoparticles, on a Thioaniline-Modified Au
Electrode
that in contrast to previous reports that incorporated enzymes into
conductive polyaniline films by physical entrapment34 or adsorp-
tion,35 the present method presents a new concept of covalently
binding the enzyme to the oligoaniline-Pt NPs composite. The
resulting Pt NPs/GOx composite was then used to probe the
biocatalytic activities of GOx. Figure 3A shows the cyclic volta-
mmograms corresponding to the analysis of glucose through the
electrochemical determination of H2O2, formed by the Pt NPs/
GOx composite electrode. In this experiment, the electrode was
prepared by the application of 60 electropolymerization cycles,
and using a molar ratio of modified Pt NPs and functionalized
GOx, in solution, that corresponded to 2.5:1.0 (for the effects of
the ratio of Pt NPs/GOx, as well as the number of electropoly-
merization cycles on the performances of the bioelectrocatalytic
electrodes, vide infra). As the concentration of glucose is elevated,
the electrocatalytic cathodic currents are enhanced, allowing the
analysis of glucose at a 1 mM limit. Figure 3A, inset, shows the
derived calibration curve. A linear relation between the current
response of the electrode and the content of glucose is observed
in the concentration range of 0-140 mM, a broad domain that
overlaps the appropriate region for analyzing sugar levels for
diabetes. The activity of the cross-linked Pt NPs/GOx composites
has been confirmed by two complementary experiments. In one
experiment, Figure 3B, it was confirmed that the electrocatalytic
reduction wave originates, indeed, from the reduction of GOx-
generated H2O2. Figure 3B, curve b, depicts the cyclic voltam-
mogram corresponding to the Pt NPs-catalyzed reduction of the
H2O2, generated by the GOx-mediated oxidation of glucose. Figure
3B, curve c, shows, however, the cyclic voltammogram of the Pt
NPs/GOx composite in the presence of glucose, upon the
coaddition of catalase to the electrolyte solution. Evidently, the
addition of catalase depleted the electrocatalytic cathodic wave,
consistent with the fact that catalase decomposes H2O2 through
a disproportionation mechanism. Further support that the enzyme
(34) Borole, D. D.; Kapadi, U. R.; Mahulikar, P. P.; Hundiwale, D. G. Polym.
Adv. Technol. 2004, 15, 306–312.
(35) Chaubey, A.; Pande, K. K.; Singh, V. S.; Malhotra, B. C. Anal. Chim. Acta
2000, 407, 907–913.
8256 Analytical Chemistry, Vol. 80, No. 21, November 1, 2008
GOx exists in a catalytically active configuration was obtained by
the activation of the bioelectrocatalytic functions of the enzyme,
in the presence of a diffusional electron mediator. The Pt NPs/
GOx-functionalized electrode was reacted with ferrocenemethanol
as a diffusional electron mediator. Electrocatalytic anodic currents
were observed in the presence of glucose, and as the concentration
of glucose increased, the catalytic currents were intensified (see
Supporting Information, Figure S3). The onset of the electrocata-
lytic anodic currents was observed at ∼0.15 V versus SCE, the
redox potential of ferrocenemethanol. These currents imply that
GOx exists in the Pt NPs/GOx composite in a biocatalytically
active structure, in which ferrocenemethanol mediates the oxida-
tion of glucose.
One aspect that should be addressed relates, however, to the
electropolymerization of the Pt NPs/GOx composite and the effect
of the electropolymerizable Pt NPs and GOx units on the
bioelectrocatalytic activity of the resulting electrode. While the
electropolymerization of the Pt NPs contributes to the 3D
conductivity of the matrix, the bioelectrocatalytic functions are
controlled by the content of the enzyme in the matrix (the H2O2
generating units) and the coverage of the Pt NPs sites. While at
first glance, it seems that high loading of the enzyme during
electropolymerization would be an advantage, due to the enhanced
biocatalytic generation of H2O2 by GOx in the matrix, the use of
a high content of enzyme in the electropolymerization mixture
would favor the incorporation of protein units around the particles,
and this would insulate the particles and prevent further growth
of the NPs/GOx film. Thus, it seems that an appropriate balance
between the electropolymerizable Pt NPs and electropolymerizable
enzyme should be retained to yield a Pt NPs/GOx composite with
optimal bioelectrocatalytic functions. Figure 4A shows the elec-
trocatalytic cathodic currents generated by Pt NPs/GOx compos-
ite electrodes, using 60 electropolymerization cycles, while
changing the ratio of electropolymerizable Pt NPs and GOx. The
concentration of the Pt NPs was kept constant (0.4 mg · mL-1)
while the concentration of GOx was varied. In this set of
experiments, the concentration of glucose was kept low, 14 mM,
Figure 3. (A) Cyclic voltammograms corresponding to the oligoa-
niline-cross-linked GOx/Pt NPs composite-modified Au electrode in
the presence of variable concentrations of glucose: (a) 0, (b) 3, (c) 7,
(d) 10, (e) 14, (f) 28, (g) 42, (h) 56, (i) 64, (j) 82, (k) 96, (l) 110, (m)
134, (n) 152, and (o) 166 mM. The electrode was interacted with the
glucose-containing electrolyte solution for a fixed time interval of 4
min prior to the recording of the respective voltammograms. Inset:
calibration curve corresponding to the electrocatalytic currents mea-
sured at E ) -0.35 V for variable concentrations of glucose. (B) Cyclic
voltammograms corresponding to the oligoaniline-cross-linked GOx/
Pt NPs composite-modified Au electrode in the presence of (a) 0 mM
glucose; (b) 64 mM glucose; (c) upon the addition of catalase, 2000
units, to a 64 mM glucose solution. Prior to the measurements, the
electrode was immersed for a fixed time interval of 4 min in the
respective electrolyte solution.
to ensure that the electrocatalytic cathodic currents are signifi-
cantly below the saturation currents. As expected, by increasing
the concentrations of GOx during the electropolymerization stage,
the bioelectrocatalytic activity of the electrode increases, and the
electrocatalytic cathodic currents reach a peak value at a concen-
tration of glucose oxidase that corresponds to 0.5 mg · mL-1.
Beyond this GOx concentration the current drops, and ultimately,
the electrocatalytic activity diminishes. This lack of bioelectro-
catalytic activity of the electrode generated at high concentrations
of GOx may be attributed to the favored electropolymerization of
the enzyme film, preventing the incorporation of the Pt NPs, or
to the rapid insulation of the electropolymerized NPs by the
enzyme, which prevents further electropolymerization and masks
the catalytic functions of the Pt NPs. The insulation of the Pt NPs
by the electropolymerized GOx seems to be particularly important,
since blocking the three-dimensional conductivity prevents, also,
Figure 4. (A) Electrocatalytic currents corresponding to oligoaniline-
cross-linked GOx/Pt NPs composite-modified Au electrode, prepared
by the application of 60 cyclic voltammetry scans between -0.1 and
1.1 V vs SCE at 100 mV · s-1, in the presence of 0.4 mg · mL-1 Pt
NPs and variable concentrations of the thioaniline-modified GOx.
Measurements were performed in a 0.1 M phosphate buffer solution
(pH 7.4) that included 14 mM glucose. The electrodes were interacted
for a fixed time interval of 4 min with the glucose-containing electrolyte
solution. The currents were extracted from the respective cyclic
voltammograms, recorded at a scan rate of 10 mV · s-1, at E ) -0.35
V. (B) Electrocatalytic currents corresponding to oligoaniline-cross-
linked GOx/Pt NPs composite-modified Au electrode, prepared by
the application of variable number of cyclic voltammetry scans
between -0.1 and 1.1 V vs SCE at 100 mV · s-1, in the presence of
0.4 mg · mL-1 Pt NPs and 0.5 mg · mL-1 GOx. Measurements were
performed in a 0.1 M phosphate buffer solution (pH 7.4) that included
56 mM glucose. The electrodes were interacted for a fixed time
interval of 4 min in the glucose-containing electrolyte solution. The
indicated currents were measured at E ) -0.35 V by cyclic
voltammetry, performed at 10 mV · s-1.
the accumulation of GOx in the resulting composite associated
with the electrode. Further support that electropolymerization at
a high GOx concentration, relative to the Pt NPs, indeed leads to
an insulation of the electrode and to the blocking of the incorpora-
tion of the Pt NPs was obtained by complementary quartz crystal
microbalance measurements. We find that electropolymerization
at a high GOx concentration leads to a sharp drop in the mass
change associated with the functionalized electrode, implying that
the formation of the Pt NPs/enzyme composite is, indeed,
perturbed. The optimal bioelectrocatalytic functions of the com-
posite electrodes were observed at a molar ratio of electropoly-
Analytical Chemistry, Vol. 80, No. 21, November 1, 2008 8257
merizable Pt NPs:GOx that corresponds to ∼2.5:1.0. Accordingly,
all of the previously described Pt NPs/GOx electrodes were
prepared using these optimal conditions.
Although the optimized ratio of thioaniline-functionalized Pt
NPs and thioaniline-modified GOx in the electropolymerization
mixture is 2.5:1.0, the question regarding the actual ratio of Pt
NPs/GOx in the cross-linked composite film must be addressed.
To characterize the composite structure associated with the
electrode we determined as a first step the content of the enzyme
in the film. For this purpose, we assayed the activity of the GOx
in the composite. Having a calibration curve that relates the
enzyme activity and the content, we concluded that the content
of GOx in the film was 2.4 × 10-7 g · cm-2. This value translates
to a coverage of 1.4 × 10-12 mol · cm-2. In the second step, we
electropolymerized the functionalized Pt NPs and the thioaniline-
modified GOx on Au quartz crystals under the optimal electropo-
lymerization conditions. By subtracting the mass of the enzyme
incorporated in the film (using the activity assay), the net weight
of the Pt NPs in the composite was estimated to be 3.5 × 10-7
g · cm-2. Knowing the size of the NPs, this value translates to a
surface coverage of 6.6 × 10-12 mol · cm-2. Thus, the molar ratio
of Pt NPs/GOx in the composite corresponds to ∼4.7:1.0.
The bioelectrocatalytic currents are controlled by the number
of electropolymerization cycles applied during the generation of
the Pt NPs/GOx electrodes in the presence of the optimal Pt NPs/
GOx ratio, Figure 4B. As the number of polymerization cycles
increases, the bioelectrocatalytic currents are intensified, and after
60 cycles, the biocatalytic currents level off to a saturation value.
The saturation value of the electrocatalytic cathodic current may
be attributed to several reasons: (i) As electropolymerization
proceeds, the three-dimensional conductivity of the Pt NPs is
perturbed by the insulating enzymes, and this eliminates the
further electropolymerization of the active components. (ii) As
polymerization proceeds, inner Pt NPs and enzyme layers become
inaccessible to glucose/H2O2, and thus, the layers do not
contribute to the total cathodic currents. Accordingly, 60 elec-
tropolymerization cycles were applied to fabricate the electrodes
for the different experiments.
The bioelectrocatalytic cathodic currents are also controlled
by the time interval allowed to interact the functionalized electrode
with glucose in the solution to yield H2O2 (see Figure S4,
Supporting Information). As the biocatalytic reaction is prolonged,
the electrocatalytic cathodic currents increase, until they level off
to a saturation value after ∼6 min. This phenomenon is explained
by the fact that H2O2 is generated in the thin enzyme film
associated with the electrode surface, and it diffuses out to the
bulk electrolyte solution, which is considered as an “empty” H2O2
reservoir. After 6 min, an equilibrium is established since the flux
of H2O2 diffusing to the bulk electrolyte is identical to the H2O2
flux generated by the enzyme film, thus leading to the saturation
of the cathodic current.
A final aspect relates to the stability of the cross-linked
composite Pt NPs/GOx electrodes toward sensing of glucose. We
find that the electrodes lose <5% in their activity upon 10-days
storage at 4 °C and lose 5-8% upon operation for 5 days in a buffer
solution at ambient temperature. This stability is remarkable since
no attempts were made to stabilize the electrodes by protective
8258 Analytical Chemistry, Vol. 80, No. 21, November 1, 2008
polymer layers or to employ sterile storage conditions or sterile
solutions.
In conclusion, the present study has introduced a new method
to fabricate integrated enzyme-Pt NPs composite films on
electrodes. The enzyme GOx catalyzed the generation of H2O2,
and the Pt NPs provided the electrocatalytic sites for the
amperometric transduction of the biocatalytic reaction (generation
of H2O2 and sensing of glucose). Interesting fundamental ques-
tions were addressed while characterizing the Pt NPs/GOx
composite. We revealed that a delicate balance between the
electropolymerizable NPs and the electropolymerizable enzyme
must be retained in order to yield an electrode with optimal
bioelectrocatalytic performance. The number of electropolymer-
ization cycles to yield the Pt NPs/GOx composite, and the time
intervals employed to interact the electrode with the glucose
solutions, were similarly found to dictate the intensity of the
amperometric responses. The high electrocatalytic cathodic cur-
rents observed at the glucose concentration range relevant for
monitoring diabetes, together with the linear current-concentration
dependence, suggest that such electrodes could be further
miniaturized into implantable glucose biosensor configurations
(for example, the determination of glucose in subcutaneous
fluids36,37). Furthermore, the preparation of integrated metal NPs/
enzyme systems cross-linked by redox-active oligoaniline bridges
suggests that direct electrical contacting of enzymes with the
electrode by means of the electroactive bridges, could be achieved.
EXPERIMENTAL SECTION
Synthesis of Pt Nanoparticles. A modified version of a
synthesis reported by Perez et al.38 was employed. A 300-mg
sample of PtCl4 was dissolved in 75 mL of hexylamine (solution
1). Then, 35 mg of thioaniline and 180 mg of 2-mercaptoethane-
sulfonic acid sodium were dissolved in 30 mL of a 1:1 methanol/
hexylamine solution (solution 2). Finally, 300 mg of sodium
borohydride was dissolved in 40 mL of a 1:1 water/methanol
solution. Following the complete dissolution of sodium borohy-
dride, hexylamine (20 mL) was added (solution 3). Solution 3 was
then poured into solution 1 under vigorous stirring at room
temperature. The reaction mixture turned brown within a few
seconds, and after 1 min, solution 2 was added to the reaction
mixture. After 3 min, 200 mL of pure water was added, and the
resulting solution was stirred for 15 min before being transferred
into a separatory funnel. Following phase separation, the water
was removed and the organic phase was repeatedly washed with
several 200-mL portions of water. The volume of the organic phase
was then reduced to ∼3-4 mL by rotary evaporation at ∼35 °C.
In the next stage, a solution containing 35 mg of thioaniline and
180 mg of 2-mercaptoethanesulfonic acid sodium salt in 15 mL
ethanol was added to the organic phase, and the resulting mixture
was stirred overnight. The black solid residue was collected by
repetitive centrifugation and subsequent washing with diethyl
(36) (a) Bindra, D.; Zhang, Y.; Wilson, G. S.; Sternberg, R.; Thevenot, D. R.;
Reach, G.; Moatti, D. Anal. Chem. 1991, 63, 1692–1696. (b) Henry, C.
Anal. Chem. 1998, 70, 594A–598A. (c) Schmidtke, D.; Freeland, A.; Heller,
A.; Bonnecaze, R. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 294–299.
(37) (a) Gross, T. M. Diabetes Technol. Ther. 2000, 2, S19–S26. (b) Feldman,
B.; Brazg, R.; Schwartz, S.; Weinstein, R. Diabetes Technol. Ther. 2003, 5,
769–779.
(38) Perez, H.; Pradeau, J.-P.; Albouy, P.-A.; Perez-Omil, J. Chem. Mater. 1999,
11, 3460–3463.
ether (3-4 times). Finally, the precipitate was separated, and it
exhibited solubility in water. A TEM analysis indicated that the
size of the modified Pt NPs corresponded to 2.0 ± 0.3 nm.
Modification of the Enzyme. GOx, 52 mg (EC 1.1.3.4 from
Aspergillus niger, 210 000 U · g-1, from Sigma) was dissolved in 3
mL of phosphate buffer solution, 0.1 M (pH 7.4). The solution
was then introduced into 52 µL of N-(maleimidocaproyloxy)
sulfosuccinimide ester (sulfo-EMCS, obtained from Pierce), 12
mg · mL-1. The resulting solution was stirred for 40 min and was
then combined with a 0.8 mL of 4-aminothiophenol (thioaniline)
solution, 1.6 mg · mL-1, in ethanol. After 2.5 h, the solution was
eluted through a G-25 column (GE Healthcare) using a phosphate
buffer, 0.1 M (pH 7.4) as the eluent. The resulting purified,
thioaniline-functionalized GOx solution was lyophilized to yield a
pale-yellow powder that was stored under -20 °C. The modified
enzyme was stable for at least three months with no noticeable
degradation in its activity.
Preparation of Modified Electrodes. Clean Au wires (0.3
cm2) were reacted for 2 h with 10 mM thioaniline solution in
ethanol. The thioaniline-modified electrodes and the thioaniline-
modified Pt NPs were subjected to the electropolymerization
process in the presence or the absence of the thioaniline-modified
GOx, using a fixed number of repetitive cyclic voltammetry scans,
ranging between -0.1 and 1.1 V versus SCE, at a scan rate of 100
mV · s-1. A phosphate buffer, 0.1 M, at pH 7.4 was used as the
electrolyte throughout the electropolymerization and test experi-
ments. All electrochemical measurements employed a PC-
controlled (Autolab GPES software) potentiostat/galvanostat
(µAutolab, type III). A graphite rod (d ) 5 mm) was used as the
counter electrode, and the reference was a saturated calomel
electrode.
Determination of the Content of GOx in the Electropoly-
merized Film. The content of the GOx in the bisaniline-cross-
linked GOx/Pt NPs-modified electrode was determined by react-
ing the electrodes with 1.5 × 10-4 M 2,2′-azino-bis(3-ethylbenzo-
thiazoline-6-sulfonic acid) (ABTS2-) and 4.2 × 10-4 M horseradish
peroxidase (1 × 106 U · g-1) and monitoring the absorbance of
the generated ABTS·- at λ ) 416 nm. A calibration curve was
derived by measuring several solutions of phosphate buffer, 0.1
M, at pH 7.4 that included different concentrations of GOx. Using
the derived calibration curve and the color generated by the Pt
NPs/GOx-cross-linked electrode, the content of the enzyme in
the matrix was elucidated.
ACKNOWLEDGMENT
Parts of this research are supported by the BIOMEDNANO
EC project.
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
Received for review July 7, 2008. Accepted August 26,
2008.
AC801398M
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