Article

pubs.acs.org/ac

Incorporating a Hybrid Urease-Carbon Nanotubes Sensitive

Nanofilm on Capacitive Field-Effect Sensors for Urea Detection
José R. Siqueira, Jr.,*,† Denise Molinnus,‡ Stefan Beging,‡ and Michael J. Schöning*,‡,§
†
Institute of Exact Sciences, Naturals and Education, Federal University of Triângulo Mineiro (UFTM), 38064-200 Uberaba, Brazil
‡
Institute of Nano- and Biotechnologies (INB), FH Aachen, Campus Jülich, 52428 Jülich, Germany
§
Peter Grünberg Institute (PGI-8), Forschungszentrum Jülich, 52425 Jülich, Germany
*
S Supporting Information

ABSTRACT: The ideal combination among biomolecules and nanomateri-

als is the key for reaching biosensing units with high sensitivity. The
challenge, however, is to find out a stable and sensitive film architecture that
can be incorporated on the sensor’s surface. In this paper, we report on the
benefits of incorporating a layer-by-layer (LbL) nanofilm of polyamidoamine
(PAMAM) dendrimer and carbon nanotubes (CNTs) on capacitive
electrolyte-insulator-semiconductor (EIS) field-effect sensors for detecting
urea. Three sensor arrangements were studied in order to investigate the
adequate film architecture, involving the LbL film with the enzyme urease: (i)
urease immobilized directly onto a bare EIS [EIS-urease] sensor; (ii) urease
atop the LbL film over the EIS [EIS-(PAMAM/CNT)-urease] sensor; and (iii) urease sandwiched between the LbL film and
another CNT layer [EIS-(PAMAM/CNT)-urease-CNT]. The surface morphology of all three urea-based EIS biosensors was
investigated by atomic force microscopy (AFM), while the biosensing abilities were studied by means of capacitance−voltage (C/
V) and dynamic constant-capacitance (ConCap) measureaments at urea concentrations ranging from 0.1 mM to 100 mM. The
EIS-urease and EIS-(PAMAM/CNT)-urease sensors showed similar sensitivity (∼18 mV/decade) and a nonregular signal
behavior as the urea concentration increased. On the other hand, the EIS-(PAMAM/CNT)-urease-CNT sensor exhibited a
superior output signal performance and higher sensitivity of about 33 mV/decade. The presence of the additional CNT layer was
decisive to achieve a urea based EIS sensor with enhanced properties. Such sensitive architecture demonstrates that the
incorporation of an adequate hybrid enzyme-nanofilm as sensing unit opens new prospects for biosensing applications using the
field-effect sensor platform.

T he manipulation of bio- and nanomaterials has permitted

the development of new sensitive hybrid nanostructures
for biosensing applications. Depending on the biosystem under
ture.2,10−13 Sensing units made by the LbL method for
detection of different biocompounds have demonstrated a
synergic effect between the biomolecules and the nanomaterial
study, specific techniques and strategies should be carefully leading to highly sensitive and selective biosensors.2,11,13
selected for the formation of such nanostructures as well as the Especially for EIS structures, the first capacitive EIS sensor
adequate choice of the transduction mode responsible for the functionalized with an LbL film containing polyamidoamine
sensor’s response signal.1−6 In particular, field-effect devices (PAMAM) dendrimer and carbon nanotubes (CNT) was
(FEDs), a special class of silicon-based sensors that are proposed with the enzyme penicillinase immobilized atop the
advantageous for sensing and biosensing, are suitable for film surface for detecting penicillin G. The use of this nanofilm
biomedical applications, exhibiting advantages in terms of as the host matrix for the penicillin immobilization generated a
miniaturized processing and reliable biological recognition at biosensor with high sensitivity toward penicillin G, also
small concentrations.7−9 The capacitive electrolyte-insulator- allowing for stable signals with low drift and fast response
semiconductor (EIS) structure is a typical class of sensors based time.14−18
on FEDs. Basically, the introduction of an additional ion- and/ In this study, we demonstrate the efficiency of using LbL
or charge-sensitive gate layer leads to changes in the electrical films made with dendrimer/carbon nanotubes as stabilizing
surface charge of the EIS surface, modulating its capacitance.7−9 matrixes for immobilization of the enzyme urease on capacitive
The key to achieve a specific biosensor with preserved EIS chips, followed by a study of the electrochemical properties
activity, high sensitivity, and selectivity rises from the employed for urea detection. This analyte is relevant in clinical diagnosis
method to immobilize an enzyme on the EIS chip. Particularly, of renal and liver diseases, with its detection being performed
the adequate combination of biomolecules and nanomaterials,
such as enzymes and carbon nanotubes, respectively, can be Received: January 24, 2014
manipulated in nanostructured layer-by-layer (LbL) films, in Accepted: May 9, 2014
which materials are disposed in a controlled nanoarchitec- Published: May 9, 2014

© 2014 American Chemical Society 5370 dx.doi.org/10.1021/ac500458s | Anal. Chem. 2014, 86, 5370−5375
Analytical Chemistry Article

frequently in medical care.19−21 The signal stability of the urea- mM polymix buffer solution. For the sandwich configuration,
based EIS sensor and the influence of carbon nanotube 200 μL of the enzyme cocktail was dropped onto the 5-bilayer
incorporation in the film structure were investigated in three PAMAM/CNT LbL nanofilm followed by an additional CNT
different arrangements: (1) atop of a bare EIS sensor, (2) atop (500 μL) layer over the immobilized urease (adsorption time =
of a 5-bilayer PAMAM/CNT nanofilm, and (3) embedded 10 min). A schematic representation of the three urea-based
between a 5-bilayer PAMAM/CNT nanofilm and an additional biosensors investigated in this study is illustrated in Scheme 1.
CNT layer.

■ EXPERIMENTAL SECTION
Materials and Solutions. Carboxylic acid functionalized
Scheme 1. Schematic Representation of the Three
Arrangements for the Urea-Based EIS Biosensors
Investigated: (A) Urease Enzyme Atop of a Bare EIS Sensor,
single-walled carbon nanotubes and fourth generation G4 (B) Urease Enzyme Atop of a 5-Bilayer PAMAM/CNT LbL
PAMAM dendrimer were purchased from Sigma-Aldrich Co. Film, and (C) Urease Enzyme between a 5-Bilayer PAMAM/
CNTs were produced via the arc technique, with mainly CNT LbL Film and an Additional CNT Layer
semiconductor features, nominal purity of 95%, length of 0.5−
1.5 μm, and diameter of 1.5−3.5 nm for individual and bundled
samples. PAMAM and CNTs solutions were prepared at a
concentration of 1.0 g L−1, using titrisol buffer solution at pH 4
and pH 8, respectively. The CNT dispersion was obtained by
adding 10 mg of the material into 10 mL of buffer solution
under ultrasonication for 2 h and then filtrated three times.
Urease enzyme (Canavalia ensiformis made of Jack beans, 50
000−100 000 units/g solid) and urea were acquired from
Sigma. The enzyme cocktail solution was prepared in
phosphate buffer solution (PBS) at pH 7 by dissolving 60 mg
of enzyme powder in 30 mL of buffer solution. Urea solutions Measurement Setup. The surface morphology of the
were prepared at different concentrations ranging from 0.1 mM PAMAM/CNT LbL nanofilms and the adhesion of the urease
to 100 mM in a 0.25 mM polymix buffer solution at pH 8, enzyme layer on the film were analyzed by an atomic force
containing 100 mM KCl solution as the ionic strength adjuster. microscope (AFM) BioMAT Workstation (JPK Instruments,
Fabrication of the EIS Sensors. The EIS structure Germany) in the tapping mode, using Si cantilevers with silicon
consisted of a p-doped ⟨100⟩ silicon substrate (356−406 μm nitride tips (8 nm radius). Images of the functionalized EIS
thick) with a specific resistance of ρ = 1−5 Ω cm. A 30 nm sensors were taken in the constant height measuring mode at
thick SiO2 insulating layer was formed via a thermal oxidation scanned areas of 1.0 × 1.0 μm2 for all arrangements, and
under O2 atmosphere at 1050 °C for 30 min. Afterward, a pH- statistical quantities were analyzed by using the Gwyddion SPM
sensitive Ta2O5 film with a thickness of ∼60 nm was deposited data analysis. The urea biosensors were electrochemically
by electron-beam evaporation of 30 nm Ta, followed by characterized by means of impedance spectra (IS), capaci-
thermal oxidation in an oxygen atmosphere at 520 °C for about tance−voltage (C/V) and constant-capacitance (ConCap)
2 h. The validation of the layer thickness was carried out by measurements using an impedance analyzer IM6 (Zahner
using an ellipsometry Nanofilm EP3 (Accurion, The Nether- Elektrik, Germany). The Bode IS was carried out in a frequency
lands). A 300 nm thick aluminum film was electron-beam range varying from 1 Hz to 1 MHz at a polarization potential of
evaporated and deposited as a rear-side contact after removing 0 V vs Ag/AgCl for determination and evaluation of a specific
the grown SiO2 on the backside of the sensor with hydrofluoric low frequency in the capacitive region of the EIS-modified
acid. As a last step, the wafer was cut into single chips of 10 × chips (see also Figure S1 in the Supporting Information). The
10 mm2 sizes with a diamond saw. The EIS chips were C/V and ConCap characterizations were performed three times
mounted into a homemade measuring cell and sealed by an O- at a frequency of 30 Hz. During the operation, a conventional
ring to protect the side walls and backside contact of the chip Ag/AgCl double-junction reference electrode (type 6.0726.100,
from solutions. The contact area of the EIS sensor with the Metrohm, Germany) with a salt bridge of 3 M KCl to set the
solution was about 0.5 cm2. working point of the EIS sensor was used. An ac voltage of 20
Fabrication of the PAMAM/CNT Nanofilms and mV was also applied to the system in order to measure the
Enzyme Immobilization. A homemade electrochemical capacitance. The contact area of the functionalized sensor
acrylic cell, sealed with an O-ring, was used as a template to surface was limited to 0.5 cm2 by the reservoir of the measuring
prepare the LbL films. The PAMAM/CNT nanofilm was cell. All measurements were performed in a dark Faraday cage
at room temperature.

■
assembled on EIS chips with the LbL technique, by alternately
dropping 1 mL of the PAMAM (5 min) and 1 mL of the single-
walled CNTs solution for 10 min each. This procedure was RESULTS AND DISCUSSION
repeated five times to generate a film with five bilayers. We Surface Morphology of the Urea-Based EIS Sensor.
adopted the strategy of assembling five bilayers based on The three arrangements of the urea-based EIS sensors were
previous reports that demonstrate the formation of highly analyzed by AFM, with the results shown in Figure 1. Height-
porous films with large surface area and without polymer traced 3D AFM images revealed agglomerates (white spots)
packing.16,17 Afterward, the film surface was rinsed with homogeneously distributed over the bare Ta2O5 surface (Figure
deionized water and dried under N2 flow. The atop-film urease 1A), which is ascribed to the immobilized urease enzyme, as
enzyme was obtained by dropping 200 μL of the enzyme demonstrated comparing this image with control images of a
cocktail onto the 5-bilayer PAMAM/CNT LbL nanofilm and bare EIS chip without enzyme presence (see also Figure S2 in
dried under room temperature, being rinsed and stored in a 0.5 the Supporting Information). Figure 1B,C shows AFM images
5371 dx.doi.org/10.1021/ac500458s | Anal. Chem. 2014, 86, 5370−5375
Analytical Chemistry Article

Figure 1. Height-traced 3D AFM images and line scans for the three urea-based EIS biosensor configurations: (A) urease enzyme atop of a bare EIS
sensor, (B) urease enzyme atop of a 5-bilayer PAMAM/CNT LbL film, and (C) urease enzyme between a 5-bilayer PAMAM/CNT LbL film and an
additional CNT layer.

of an enzyme layer atop of a 5-bilayer PAMAM/CNT nanofilm Electrochemical Characterization and Urea Detection.
and sandwiched between the LbL film and an additional CNT The biosensing ability toward urea of the modified EIS sensors
layer, respectively. Figure 1B exhibited clearly the spherically was investigated by means of C/V measurements at urea
shaped urease randomly and homogeneously covering the concentrations ranging from 0.1 mM to 100 mM. Figure 2
PAMAM/CNT film surface, while Figure 1C displays the
presence of the additional CNT layer over the enzyme. Both
images demonstrate the interpenetration of CNTs and enzyme
molecules in a highly porous fashion with a large surface area.
This latter is supported by line scans of each image that exhibits
the increasing of the height profile among the three
arrangements and is confirmed by the mean roughness (rms)
of each sensor, as shown in Table 1. As expected, the sensor

Table 1. Mean Roughness (rms) Exhibited by the Three

Urea-Based EIS Biosensor Arrangements
sensor configuration rms roughness (nm)
EIS-urease 17.1
EIS-(PAMAM/CNT)-urease 18.8
EIS-(PAMAM/CNT)-urease-CNT 20.8

Figure 2. C/V curves at urea concentrations ranging from 0.1 mM to

EIS-(PAMAM/CNT)-urease-CNT presented the higher height
100 mM for the arrangement with the enzyme atop the PAMAM/
profile, leading to a higher roughness (rms) and larger surface CNT LbL film. Inset: shift of the bias voltage to higher values with
area, demonstrating the influence of the nanofilm architecture increasing urea concentrations at the sensor surface due to the
on the morphological properties. The interconnection of enzymatic reaction.
nanotubes into the film can act as fast ionic-conducting
channels, while the larger surface area permits a higher amount presents results for the arrangement with the enzyme atop the
of enzyme immobilization and better distribution over the PAMAM/CNT LbL film. The region between −1.0 and 0.0 V
sensor surface. These features are desirable for sensor-based corresponds to the depletion zone that is intrinsic to the
EIS structures, leading to enhanced sensing properties semiconductor EIS structure (p-Si−SiO2−Ta2O5). Changes in
including stable signals and high sensitivity. this region indicate changes at the solid/liquid interface
Note that the same amount of enzyme was immobilized for potential, thus modulating the flat-band voltage of the EIS
all three configurations. The arrangements containing the sensor. In particular, the principle based on EIS sensors for urea
sensitive nanofilm (LbL-enzyme) were stable against several detection consists of the enzymatic reaction,19−21 as depicted in
rinsing processes with aqueous solutions. On the other hand, eq 1:
the same did not occur for the bare EIS-enzyme arrangement,
urease
presenting a poor mechanical stability over the chip. This (NH 2)2 CO + 3H 2O ⎯⎯⎯⎯⎯→ 2NH4 + + OH− + HCO3−
contrast confirms the suitability of using the PAMAM/CNT (1)
LbL film as host matrix for enzyme immobilization. Moreover,
we have found in subsidiary experiments that other film- The zoomed area around 32 nF in the inset shows the shift
forming techniques, viz., casting or spin-coating, could not be of the bias voltage to higher values with increasing urea
used, as the resulting films were not as homogeneous and stable concentrations, which indicates an increased OH−-ion concen-
as for the LbL films. tration at the sensor surface due to the enzymatic reaction. It is
5372 dx.doi.org/10.1021/ac500458s | Anal. Chem. 2014, 86, 5370−5375
Analytical Chemistry Article

stressed that the same behavior in the C/V curves was also
observed for bare EIS-urease and EIS-(PAMAM/CNT)-urease-
CNT sensors. The schematic illustration of the measurement
setup and the operation principle of the urea biosensor-based
EIS structure modified with a matrix film of PAMAM/CNT
LbL film as well as the schematic representation of enzymatic
reaction are depicted in Scheme 2.

Scheme 2. Scheme of the Measurement Setup Illustrating the

Operation Principle and the Chemical Reaction for a Urea
Biosensor-Based EIS Structure Modified with a Sensitive
PAMAM/CNT LbL Nanofilm and the Enzyme Urease

Furthermore, changes among the three sensors were

observed in the maximum capacitance (CMAX) behavior (see
also Figure S3 in the Supporting Information). While the CMAX
values for the EIS-urease and EIS-(PAMAM/CNT)-urease
sensors were recorded at approximately 48.0 nF and 47.0 nF,
respectively, for the EIS-(PAMAM/CNT)-urease-CNT sensor
a CMAX value of around 44.0 nF was recorded. This implies that
the overall capacitance of the EIS structure is decreasing with
increasing layer thickness, which agrees with the theoretically
expected behavior. The differences for each arrangement are Figure 3. Dynamic ConCap responses for urea concentrations ranging
related to the presence of additional layers on the EIS surface from 0.1 mM to 100 mM for bare EIS-urease (A), EIS-(PAMAM/
(LbL film, enzyme, and CNT). In terms of equivalent circuits, CNT)-urease (B) and EIS-(PAMAM/CNT)-urease-CNT (C) sen-
each arrangement corresponds to an additional parallel sors, and their corresponding calibration curves (D).
capacitor associated with the capacitive EIS structure.7−9,22−24
In order to investigate in detail the difference on the sensitive
characteristics and to evaluate the biosensing ability of the sensors, the output signal increased somewhat irregularly after
modified urea-based EIS biosensors toward urea, ConCap urea additions, exhibiting a sensitivity of 18 mV/decade (R =
measurements were further performed using the same range of 0.996) and 18.5 mV/decade (R = 0.992) (Figure 3D) and a
urea concentrations (0.1 mM−100 mM). Figure 3 presents sensor drift of 0.2 mV/min and 1.1 mV/min, respectively.
dynamic ConCap responses of urea concentrations for the bare In contrast, the EIS-(PAMAM/CNT)-urease-CNT sensor
EIS-urease (A), EIS-(PAMAM/CNT)-urease (B), and EIS- exhibited a linear output signal performance and higher
(PAMAM/CNT)-urease-CNT (C) sensor, respectively, as well sensitivity. Figure 3C shows clearly the constant increase of
as their corresponding calibration curves (D). The increase in each potential step from lower to higher urea concentrations
OH−-ion concentration from the enzymatic reaction (owing to without saturation. Such enhancement in the detection signal
the catalyzed hydrolysis of urea into ammonium ions) in the led to a sensor with superior sensitivity toward urea of 33 mV/
presence of higher-concentrated urea solutions was determined decade (R = 0.999). This value is almost 2-fold higher than for
from changes in the depletion potential at a fixed working point the other two sensor arrangements analyzed, as demonstrated
value of 32 nF. This capacitance value equals the flat-band in the calibration curve in Figure 3D. The stable detection
region of the EIS field-effect sensor (see Figure 2). This region signal, with a low drift of 0.5 mV/min, followed with the
is typically selected in field-effect devices to study the influence sensitivity achieved may envisage the application of this sensor
of concentration changes of the analyte at the interface sensor/ arrangement to detect urea concentrations in human samples,
liquid, finally delivering a detectable electrochemical potential. like blood or urine.
All the sensors were sensitive for a low urea concentration of For urea concentrations higher than 50 mM, the sensor
0.1 mM. Considering the best linear range, viz., from 5 mM to signal enters into a drift regime and saturates for the three
100 mM, both the bare EIS-urease and the EIS-(PAMAM/ sensors configurations. This is a typical behavior for
CNT)-urease sensors showed a similar signal response and potentiometric enzyme sensors for higher-concentrated analy-
sensitivity toward different urea concentrations. For these tes. On the one hand, it indicates that the urea concentration is
5373 dx.doi.org/10.1021/ac500458s | Anal. Chem. 2014, 86, 5370−5375
Analytical Chemistry Article

too high to be hydrolyzed completely by the urease enzyme, sensors, without enzyme immobilization, were submitted to
needing more time to stabilize the sensor output signal. On the urea detection. Once all the urea solutions were at the same
other hand, at high urea concentrations, the pH change due to adjusted pH value, no signal changes were observed.
hydrolysis at the interface enzyme membrane/sensor surface is The enhanced performance of the urea biosensor with the
such strong that it additionally (negatively) influences the urease between the LbL film and the additional CNT layer may
enzyme activity as well. These are two opposing effects. be explained based on a previous study, which correlates the
However, at 100 mM urea concentration, one may note a signal morphological and chemical changes induced by the presence
with lower drift for the EIS-(PAMAM/CNT)-urease-CNT of carbon nanotubes on the sensitive film structure containing
sensor, leading to a faster stable response time. This might be the enzyme urease.32 It is already known that the combination
attributed due to the fact that this sandwich-type, layered dendrimer-nanotubes results in a host matrix nanofilm with a
structure serves as a kind of diffusion barrier somewhat limiting larger active surface area, which can allow both a better
the amount of substrate molecules at higher urea concen- distribution and attachment of a higher amount of enzyme
trations and, thus, extending the linear measurement range of molecules immobilized on its surface.15−17 The intimate
the biosensor and demonstrating that the additional CNT layer contact between PAMAM and dense network bundles of
incorporated atop the enzyme advantageously affects the CNTs form stable multilayers highly interconnected among
electrochemical properties of the sensor. them, resulting in a highly porous film. This porosity may offer
A direct comparison with urea-based EIS sensors in the easier ion penetration (in our case OH−-ions, see Scheme 2)
literature is difficult because diverse parameters such as the from the enzymatic reaction onto the EIS surface. In the
oxide layer employed as pH-sensitive ionic layer, supporting particular case, the additional nanotube layer when connected
electrolyte, active area, and signal frequency are different. In with the urease may lead to an enhancement of the enzyme
addition, one has to consider the influence of membranes or activity due to fact that the enzyme becomes more stable into
nanofilms, which can modify the pH-sensitive behavior of the the film, causing a better molecular accommodation of the
semiconductor transducer layer. Nevertheless, the performance enzyme and facilitating the access of the catalytic substrate
of the EIS-(PAMAM/CNT)-urease-CNT, in terms of its (urea). The assembled CNTs layer can interact with the LbL
output signal response, sensitivity, and detection limit, is film and also with the enzyme layer in a noncovalent nature
superior or comparable to recent urea-based EIS biosensors, (van der Waals and electrostatic forces), permitting that CNTs
thus demonstrating the suitability of employing specific hybrid interpenetrate into the urease layer and interact directly on the
nanofilm-based nanotubes as stabilizer matrix for enzyme LbL film.32 Therefore, the embedded nanotube layer atop the
immobilization. For instance, Pan et al. introduced EIS devices urease enzyme may act as a scaffold that stabilizes the enzyme
employing diverse sensing membranes deposited on Si molecules onto the EIS chip, preserving its catalytic sites, and
substrates by means of different physical deposition methods thus enhancing the sensor performance with increasing
and applied them as urea biosensor. EIS devices with sensing sensitivity and signal response for urea detection.

■
membranes of Nd2TiO5 and TiO2/Er2O3 as gate insulator
membrane achieved a sensitivity around 9.0−10.0 mV/mM in a
linear range of urea concentrations about 3−40 mM.25,26 In
CONCLUSIONS
another report, a urea-based EIS sensor with a sensing In summary, we demonstrated that the use of the PAMAM/
membrane of Sm2O3 exhibited a sensitivity of 2.45 mV/mM CNT LbL film as a matrix for urease immobilization with an
at a concentration range of 5−40 mM.27 An EIS-based urea additionally deposited nanotube layer atop the enzyme was
biosensor incorporating a PrYxOy sensing film presented a decisive to achieve a urea-based EIS sensor with enhanced
sensitivity of 9.59 mV/mM at a concentration range of 1−16 properties. In comparison to the EIS-urease and the EIS-
mM.28 A sensitivity of 72.85 mV/purea at a concentration range (PAMAM/CNT)-urease sensors, the EIS-(PAMAM/CNT)-
of 0.1−32 mM was achieved for an EIS structure with a sensing urease-CNT sensor exhibited an improved signal response and
layer of Sm2TiO5.29 In the same concentration range, another higher sensitivity assigned to the appropriate nanofilm
report showed an EIS sensor with a novel high-k Dy2TiO5 structure, which created an adequate environment to allocate
sensing membrane with a sensitivity of 118.38 mV/pC.30 In and to preserve the active sites of the enzyme urease on the
contrast to all these examples, the proposed Al/Si/SiO2/Ta2O5 sensor surface. An important implication of our findings is
EIS structure has the advantage that the fabrication process is related to the possible use of this urea biosensor in real clinical
fully compatible with silicon planar technology that enables a assays, such as the determination of urine with high
cost-effective production with regards to future practical concentrations of interfering ions and lithogenic substances
applications.31 that usually can disturb the urea detection. Furthermore, to our
Regarding electrochemical film stability, the arrangement knowledge, this is the first report on the utilization of such
LbL film-urease and LbL film-urease-CNT were stable on the hybrid urease-carbon nanotube sensitive nanofilm for the
EIS chip during the characterization period for urea detection. detection of urea based on capacitive EIS sensors. We proved
In contrast, a weak stability was observed with urease that the use of a PAMAM/CNT LbL film as host matrix for
incorporated directly on the bare EIS surface, in which the enzyme immobilization is entirely generic and may be extended
enzyme layer starts to degrade after repeating the set of to other enzymes, opening strategies for new capacitive EIS
measurements for urea detection. Also worth mentioning was biosensors with superior and optimized performance.

■
the selectivity toward urea of the sensor arrangements. In the
course of experiments, highly concentrated glucose (50 mM) ASSOCIATED CONTENT
and polymix buffer solutions were alternately added with the
urea solutions, but no signal changes were noted with either *
S Supporting Information

glucose or polymix buffer solutions. No signal response was Additional information as noted in text. This material is
also observed when bare EIS and EIS-(PAMAM/CNT) available free of charge via the Internet at http://pubs.acs.org.
5374 dx.doi.org/10.1021/ac500458s | Anal. Chem. 2014, 86, 5370−5375
Analytical Chemistry

■
Article

AUTHOR INFORMATION (25) Pan, T.-M.; Lin, J-.C.; Wu, M.-H.; Lai, C.-S. Biosens. Bioelectron.
2009, 24, 2864−2870.
Corresponding Authors
(26) Pan, T.-M.; Lin, J-.C. Sens. Actuators, B 2009, 138, 474−479.
*E-mail: jr.siqueira@fisica.uftm.edu.br. Phone: +55 34 3316 (27) Wu, M.-H.; Cheng, C.-H.; Lai, A.-S.; Pan, T.-M. Sens. Actuators,
1818. B 2009, 138, 221−227.
*E-mail: [email protected]. Phone: +49 241 6009 (28) Wu, M.-H.; Lee, C.-D.; Pan, T.-M. Anal. Chim. Acta 2009, 651,
53215. 36−41.
Notes (29) Pan, T.-M.; Huang, M.-D.; Lin, W.-Y.; Wu, M.-H. Anal. Chim.
The authors declare no competing financial interest. Acta 2010, 669, 68−74.

■
(30) Pan, T.-M.; Lin, C.-W. J. Electrochem. Soc. 2011, 158, J100−J105.
ACKNOWLEDGMENTS (31) Schöning, M. J.; Brinkmann, D.; Rolka, D.; Demuth, C.;
Poghossian, A. Sens. Actuators, B 2005, 111−112, 423−429.
The authors are grateful to the Brazilian Foundations CNPq (32) Caseli, L.; Siqueira, J. R., Jr. Langmuir 2012, 28, 5398−5403.
(Grant 480400/2010-5), FAPEMIG (Grant APQ-01064-12),
and nBioNet network (CAPES) for the financial support.
Furthermore, the authors gratefully thank Matthias Bäcker and
Heiko Spelthahn for technical support.

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