Nanomolar Detection of Glutamate at a Biosensor Based on Screen-Printed Electrodes Modified with Carbon Nanotubes

Abstract

The amperometric glutamate biosensor based on screen-printed electrodes containing carbon nanotubes (CNT), and its integration in a flow injection analysis system, is described herein. The sensor was fabricated by simply adsorbing enzyme glutamate oxidase (GlutOx) on a commercial substrate containing multi-wall CNT. The resulting device displayed excellent electroanalytical properties toward the determination of L-glutamate in a wide linear range (0.01-10 μM) with low detection limit (10 nM, S/N≥3), fast response time (≤5 s), and good operational and long-term stability. The CNT modified screen-printed electrodes have a potential to be of general interest for designing of electrochemical sensors and biosensors.

Full text

Nanomolar Detection of Glutamate at a Biosensor Based on
Screen-Printed Electrodes Modified with Carbon Nanotubes
Raju Khan,a, b Waldemar Gorski,bCarlos D. Garcia*b
aAnalytical Chemistry Division, CSIR-North East Institute of Science & Technology, Jorhat, 785006, Assam, India
bDepartment of Chemistry, The University of Texas at San Antonio, One UTSA Circle, San Antonio, TX, 78249, USA
phone: 01 (210)458-5774, fax: 01 (210) 458-5428
*e-mail: carlos.garcia@utsa.edu
Received: July 2, 2011;
&
Accepted: August 15, 2011
Abstract
The flow injection analysis (FIA) of monosodium l-glutamate (MSG) was performed electrochemically using a bio-
sensor based on screen-printed electrodes containing carbon nanotubes (CNT). The sensor was fabricated by simply
adsorbing glutamate oxidase (GlutOx) on the electrode surface. The resulting device displayed excellent electroana-
lytical properties toward the determination of l-glutamate in a wide linear range (0.01–10 mM) with low detection
limit (10 nM, S/N3), fast response time (5 s), and good operational and long-term stability. The CNT-modified
screen-printed electrodes have a potential to be of general interest for easy preparation of electrochemical sensors
and biosensors relevant for biomedical applications.
Keywords: Biosensors, l-Glutamate, Glutamate oxidase, Flow injection analysis, Carbon nanotubes
DOI: 10.1002/elan.201100348
1 Introduction
Biosensors have been applied in many fields including
clinical diagnostics, food processing, and biomedical re-
search in order to quantify the selected chemical species.
Among others, the analysis of l-glutamate has received
much attention recently. l-glutamate (Glut) is one of the
most commonly found amino acids in nature [1] and has
critical neurological functions including neurotransmis-
sion [2,3]. Monosodium l-glutamate (MSG) is also an im-
portant flavor enhancer, which is typically added to the
processed meats, poultry, seafood, snacks, soups, and
stews at a concentration ranging from 0.1 to 0.8 wt% [4] .
Although most regulatory agencies have affirmed the
safety of MSG, at levels normally consumed by the gener-
al population, many customers have the perception that
glutamate may have detrimental health effects [4]. In re-
sponse to such perception, and due to the important bio-
logical functions of l-glutamate, various instrumental
methods based on liquid chromatography have been de-
veloped for its analysis [5, 6] . Several alternative ap-
proaches have also been developed to quantify the l-glu-
tamate including methods based on capillary electropho-
resis [7], chemiluminometry [8], fluorescense [9], and
electrochemistry. Although the electrochemical detection
of l-glutamate is typically based on the use of enzymes
immobilized in reactors [10], microdialysis probes [11],
nanocomposites [12], or on gold surfaces [13], the most
common detection technique is amperometry [14–18].
Some of the reasons for this trend include its simple in-
strumental setup, potential for miniaturization, sensitivity,
and fast response.
Among other carbon-based materials, carbon nano-
tubes (CNT) are one of the preferred substrates for the
design of biosensors [14,15]. Among other advantages it
is worth highlighting high surface area [16] and the possi-
bility to enhance the electrochemical reactivity of differ-
ent biomolecules [14,17–20].
The present paper describes the amperometric biosen-
sor for the flow injection analysis (FIA) at nM levels. The
l-glutamate biosensor was prepared by adsorption of the
enzyme l-glutamate oxidase (GlutOx) on a commercial
screen-printed substrate containing CNT. In addition to
providing a robust platform that integrates the working,
counter, and reference electrodes, the selected substrate
could be easily integrated in the FIA system, which facili-
tated sample-handling operations and improved the over-
all throughput of the analytical method. The following
sections provide the characterization of the l-glutamate
biosensor and the optimization of experimental condi-
tions in order to maximize its signal. We also demonstrate
the use of such sensor for the analysis of glutamate in a
real sample (spiked soy sauce).
2 Experimental
2.1 Molecular Modeling
Molecular modeling calculations were performed in order
to probe the interactions between the enzyme molecules
Electroanalysis 2011, 23, No. 10, 2357– 2363  2011 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim 2357
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and carbon nanotubes. The docking of a GlutOx molecule
to a 5-nm long single-wall carbon nanotube (Nanotube
Modeler V 1.7.1, JCrystalSoft) was modeled using Auto-
Dock Vina 4.2 [21] and the X-ray crystal structure of the
enzyme (PDB ID: 2E1 M) [22]. The grid box with dimen-
sions of 100  in the X,Y, and Zdirections was used
with its center placed at the center of the enzyme mole-
cule. This grid size was selected in order to include the
entire enzyme molecule and, thus, allowing the CNT to
randomly interact with the whole surface of the protein.
The spacing was set at 0.653 , which defined 1,030,301
points for analysis. The calculations did not include the
solvent effects or post-adsorption structural rearrange-
ments of the enzyme. Out of the nine potential docking
sites identified by the model, the preferred configuration
was defined as the one with the minimum potential
energy and maximum number of poses clustered at that
site. The calculations lasted less than 5 h when performed
on a MacBook Air (2.13 GHz Intel core duo processor,
4GB RAM memory, and Leopard 10.6.7). The graphical
analysis of the results was performed by using the PyMol
molecular visualization program [23].
2.2 Regents and Solutions
The aqueous solutions were prepared by using analytical
grade regents and ultrapure water (18 MWcm1, NANO-
pure Diamond; Barnstead, Dubuque, IA). l-glutamate
oxidase (from Streptomyces sp., 5.0 U.mg1), H2O2,
sodium l-glutamate, l-cysteine, acetaminophen, and l-as-
corbic acid were purchased from Sigma–Aldrich (Saint
Lois, MO). Other chemicals (NaH2PO4·H2O, Na2HPO4,
HCl, and NaOH) were purchased from Fisher Scientific
(Fairlawn, NJ). Sodium phosphate buffer solution
(0.10 M, pH 7.40) was prepared daily and used as a back-
ground electrolyte for the electrochemical detection as
well as a carrier solution in the flow injection system.
Stock solutions of glutamate (0.100 M) were prepared in
phosphate buffer solutions. All experiments were per-
formed at room temperature (2218C).
2.3 Electrochemical Measurements
A CHI-810B workstation (CH Instruments; Austin, TX)
was used to characterize the biosensors and to perform
the electrochemical detection experiments. In all cases,
the commercial screen-printed electrode substrates
(DRP-110CNT, DropSens; Asturias, Spain) with 4.0-mm
dia. multiwall CNT-COOH working electrode, carbon ink
counter electrode, and an integrated silver pseudo-refer-
ence electrode were used. The pseudo-reference elec-
trode had a potential of +20 mV vs. conventional Ag/
AgCl/NaCl 3 M reference electrode when measured in
0.10 M phosphate buffer (pH 7.40).
2.4 Biosensor Preparation
In order to prepare the biosensors, 10.0 mL of a freshly
prepared solution of l-glutamate oxidase (5.0 U mL1)
was cast on the working electrode and incubated at 4 8C
overnight. The conditions for the immobilization of the
enzyme were selected based on prior studies from our
group [24–27]. The biosensors were rinsed with a buffer
solution to remove loosely-bound material and stored at
48C in pH 7.40 phosphate buffer solution when not in
use.
2.5 Flow Injection Analysis
The FIA experiments were carried out by using a home-
made flow system that comprised a 12-cylinder peristaltic
pump (Gilson Minipuls 3; Middleton, WI), manual six-
port rotary injection valve (Rheodyne 9725; Rohnert
Park, CA), and wall-jet flow cell (DropSens ; Asturias,
Spain) placed downstream. The injection valve included a
20-mL sample loop (PEEK tubing, 0.020“ ID 1/16” OD 
10.1 cm, Upchurch Scientific; Oak Harbor, WA). The
DropSens wall-jet cell for FIA was composed of two
transparent methacrylate blocks with the inlet and outlet
flow channels that impinged the carrier solution on the
detection electrode. The open-close system (no screws
needed) of the flow cell allowed for easy sensor replace-
ment. After the modification of the working electrode
with the enzyme solution, the screen-printed electrode
substrate was placed in between the cell blocks and con-
nected to the potentiostat using an ad-hoc connector. The
system was flushed with the carrier solution to remove
the bubbles and the baseline current was recorded. Once
the baseline was stabilized (<5 min), the solutions con-
taining the selected analyte (l-glutamate or hydrogen per-
oxide) were injected into the flow stream and the FIA-
gram was recorded at a selected constant potential. The
data points and error bars presented in this manuscript
represent the average peak currents and standard devia-
tions, respectively, of at least three consecutive injections.
3 Results and Discussion
3.1 Molecular Modeling Studies
The AutoDock Vina molecular docking program was
used to identify the most likely mode by which the
enzyme molecule adsorbs to a single carbon nanotube.
Figure 1 shows that the identified docking site involved
the interactions of CNT with random coil structures of
the enzyme. The overall calculated binding energy was
equal to 28.0 kcal mol1, which indicated that the ad-
sorption was sufficient to immobilize the GlutOx on the
surface of CNT. In addition, the molecular visualization
indicated that the adsorption of enzyme molecules on
carbon nanotubes should not affect the accessibility of
the active sites (Argyr124, Arg305, His312, Gly316,
2358 www.electroanalysis.wiley-vch.de  2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2011, 23, No. 10, 2357 – 2363
Full Paper R. Khan et al.

Tyr545, Tyr562, Trp564, and Trp653) [22] to l-glutamate
molecules.
3.2 Substrate Characterization
The surface of the working electrodes on the substrates
was investigated by scanning electron microscopy (SEM).
Figure 2 shows that the working electrode was made of a
highly porous matrix with embedded CNT. Such CNT-
based matrices have shown good capacity for enzyme im-
mobilization [28,29] and electrooxidation of hydrogen
peroxide produced by enzymatic reactions [30]. The ex-
tended network of nanopores results in the increased
roughness of the electrode surface, which leads to the in-
creased active surface area and better sensitivity of such
devices [16]. These features in conjunction with the low
cost and robustness of such electrodes are very attractive
in the development of sensitive biosensors.
The voltammetric properties of the plain electrodes
were investigated by recording cyclic voltammograms in a
pH 7.40 phosphate buffer solution (data not shown). The
voltammograms were featureless in a wide potential
window (from 0.5 to 1.2 V vs. the Ag pseudo-reference
electrode) and revealed relatively low background cur-
rents (<0.6 mA).
3.3 Selection of Detection Potential
The H2O2produced in the enzymatic reactions of oxidas-
es can be detected electrochemically under a variety of
conditions [31,32]. One of the key parameters in the op-
eration of electrochemical biosensors is the potential ap-
plied to a working electrode. Higher detection potentials
typically yield higher signals (peak currents) but they typ-
ically result in poorer selectivity and longer baseline sta-
bilization time. In order to determine the optimum detec-
tion potential, a hydrodynamic voltammogram was ob-
tained by performing sequential injections of 10.0-mM
glutamate aliquots into a flowing solution and changing
the applied potential in the 0.60–1.20 V range. Figure 3
summarizes the relationship between the analyte peak
current and the potential applied to the working elec-
trode for both the l-glutamate and hydrogen peroxide.
Figure 3 shows that the biosensor did not respond to
the injection of either l-glutamate or H2O2at potentials
lower than 0.7 V. At higher potentials, the parallel in-
crease in the peak current for both the l-glutamate and
H2O2was observed. Such behavior supports the notion
that the glutamate detection was via the oxidation of
H2O2that was produced in the enzymatic Reaction 1.
Fig. 1. Binding pose of GlutOx on the CNT according to the AutoDock Vina molecular docking program. The amino acids of the en-
zymes active site are indicated by arrows.
Electroanalysis 2011, 23, No. 10, 2357– 2363  2011 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim www.electroanalysis.wiley-vch.de 2359
Nanomolar Detection of Glutamate

l-glutamate þO2þH2O2GlutOx
!
l-ketoglutarate þH2O2þNH3ð1Þ
In order to preserve practical sensitivity and avoid
longer baseline stabilization times at high positive poten-
tials, a detection potential of 0.95 V was selected and
used for the rest of the experiments described in this
manuscript. Although the oxidation of hydrogen peroxide
at 0.95 V proved to be a simple working approach to the
signal transduction in the proposed biosensor (vide infra),
the use of high detection potentials can limit the selectivi-
ty of the sensor. In such cases, the alternative option
would be to use the low-potential reduction of hydrogen
peroxide catalyzed by horseradish peroxidase [33]. How-
ever, that alternative would require the incorporation of
a second enzyme into the biosensor [34, 35] .
The stability of the signal at 0.95 V was examined by
performing consecutive injections of 10.0 mMH
2O2(data
not shown). Only negligible changes in peak current were
observed, which indicated the relevance of screen-printed
electrodes as suitable substrates for the development of
electrochemical biosensors.
3.4 Selection of Flow Rate
The effect of flow rate on the response of the biosensor
was studied by injecting 10.0-mM aliquots of l-glutamate
in the flow injection analysis system.
Figure 4 shows that the peak current due to the l-gluta-
mate injection was higher at lower flow rates. This can be
explained by considering that at low flow rates the resi-
dence time of l-glutamate in the detection cell is longer,
which allows for the generation of more hydrogen perox-
ide (as shown in Reaction 1). This, in turn, leads to larger
peak current due to the electro-oxidation of hydrogen
peroxide. However, the application of very low flow rates
negatively affected the widths of the injection peaks (e.g.
they increased from 91 s at 6.0 mL min1to 351s at
1mL min
1), which ultimately determine the throughput
of the analytical technique. In order to preserve a good
signal-to-noise ratio along with the acceptable sampling
frequency (up to 7 samples min1), the flow rate of
3.0 mL min1was selected as optimal and used in all sub-
sequent experiments.
3.5 Selection of pH
The effect of pH on the peak current was investigated in
the pH range from 6.6 to 8.0 by injecting 10.0-mM aliquots
of glutamate solution into the carrier solution. Outside of
Fig. 2. SEM micrograph of the surface of screen printed elec-
trodes containing multiwall CNT.
Fig. 3. Hydrodynamic voltammograms of (a) 10 mMl-Gluta-
mate, and (b) 10 mMH
2O2recorded at the biosensor. Carrier so-
lution, pH 7.40 phosphate buffer (0.10 M). Flow rate, 3.0 mL
min1.
Fig. 4. The effect of flow rate on the current response of the
biosensor to (a) 10.0 mMl-glutamate, and (b) 10 mMH
2O2. Car-
rier solution, pH 7.40 phosphate buffer (0.10 M). Potential,
0.95 V.
2360 www.electroanalysis.wiley-vch.de  2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2011, 23, No. 10, 2357 – 2363
Full Paper R. Khan et al.

this pH range, a significant enzyme denaturing was ob-
served in accordance with the previous reports [36].
Figure 5 shows a bell-shaped current – pH curve with a
maximum response in the pH range from 7.2 to 7.6. This
range includes the optimum pH 7.4, which was reported
for the free GlutOx enzyme [36]. This suggests that the
enzymatic reaction rather than the electrochemical oxida-
tion of H2O2determines the response of the biosensor to
l-glutamate. In order to maximize the biosensors signal,
a phosphate buffer solution (pH 7.4) was selected as the
optimum carrier solution for all further experiments.
3.6 Analytical Figures of Merit
Under the optimized experimental conditions, the linear
relationship between the l-glutamate concentration and
the biosensors signal extended over the three orders of
magnitude (0.01–10 mM). The sensitivity of the detection,
as defined by the slope of the linear range of the calibra-
tion curve, was equal to 0.720.05 mAmM1(R=0.970).
The reproducibility of the signal was evaluated by record-
ing the peak current due to the injection of 10.0 mMl-
glutamate solution into the FIA system. The relative stan-
dard deviation of the average peak current was below
6% (N=50). In order to examine the long-term storage
stability, the response of the biosensor was examined by
performing 20 consecutive injections of 10.0 mMl-gluta-
mate aliquots every 3 days during a 24-day test period.
The biosensor was rinsed with a buffer solution and
stored in a pH 7.40 phosphate buffer solution at 48C
when not in use. Figure 6 shows that the biosensor re-
tained 92% of its initial signal after 24 days. This docu-
ments a good long-term stability of the glutamate oxidase
in the proposed biosensor. This also corroborates prior
reports stating that the adsorption of enzymes on the
CNT is predominantly irreversible [2, 16, 24–26] and that
some of the enzyme adsorbed on the CNT can undergo
deactivation.
The biosensors response to some of the most common
potential interferences (ascorbic acid, l-cysteine, and
acetaminophen) was also investigated. Figure 7 shows
that at a concentration level of 100 mM (10 times higher
than that of l-glutamate) the interference level of the
three species was approximately 3 %. It should be pointed
out that we recorded a high level of interference from l-
cysteine and acetaminophen but at alkaline pH values
(pH>10, data not shown), which is in agreement with
previous reports [28].
Fig. 5. Effect of pH on the amperometric response of the bio-
sensor to 10.0 mMl-glutamate. Carrier solution, pH 7.40 phos-
phate buffer (0.10 M). Flow rate, 3.0 mL min1. Potential, 0.95 V.
Fig. 6. Shelf-stability of the biosensor measured by sequential
injections of 10.0 mMl-glutamate solution into a carrier solution.
Carrier solution, pH 7.40 phosphate buffer (0.10 M). Flow rate,
3.0 mL min1. Potential, 0.95 V.
Fig. 7. Amperometric trace recorded at the biosensor for injec-
tions of (a) 100 mM ascorbic acid, (b) 100 mMl-cysteine, (c)
100 mM acetaminophen, and (d) 10 mMl-glutamate. Carrier solu-
tion, pH 7.40 phosphate buffer (0.10 M). Flow rate, 3.0 mL
min1. Potential, 0.95 V.
Electroanalysis 2011, 23, No. 10, 2357– 2363  2011 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim www.electroanalysis.wiley-vch.de 2361
Nanomolar Detection of Glutamate

3.7 Determination of l-Glutamate in Real Samples
The biosensor was used to determine the l-glutamate in a
real sample of soy sauce. The sample, that contained no
MSG, was spiked to a final concentration of 10 mMl-glu-
tamate and analyzed by the standard addition method
using the optimized conditions. This method was selected
because of the presence of other electrochemically-active
compounds in the as-received sample. The analysis yield-
ed a concentration of 9.80.1 mM(N=3), which illustrat-
ed the merit of the proposed biosensor for the determina-
tion of l-glutamate in the matrix of soy sauce.
3.8 Comparison with Other Electrochemical l-Glutamate
Biosensors
Table 1 shows a comparison of our biosensor with other
relevant glutamate biosensors published in open litera-
ture. The proposed new biosensor displays a wide linear
range of three orders of magnitude, which is comparable
to that of the other biosensors. However, it displays a
very low detection limit (10 nM), which is among the best
reported thus far. Other advantageous features of the
proposed biosensor include the use of a commercially-
available electrode substrate combined with the simplicity
of enzyme immobilization.
4 Conclusions
The results described in this paper demonstrate the feasi-
bility of fabricating biosensors by simply adsorbing en-
zymes on a commercially-available screen-printed sub-
strate modified with carbon nanotubes. Such approach
allows for the development of reliable sensors with detec-
tion limits in the nanomolar range. The integration of
such biosensors with flow injection analysis systems facili-
tates the sampling and handling operations, therefore im-
proving the throughput of the method.
Acknowledgements
Financial support for this project was provided by the
Welch Foundation Departmental Research Grant (AX-
0026), National Institutes of Health through the National
Institute of General Medical Sciences (1SC3GM081085),
and the Research Centers at Minority Institutions
(2G12RR013646-11). Dr. R. Khan is thankful to the De-
partment of Science & Technology (DST), Government of
India for financial support received under the BOY-
SCAST Fellowship No. SR/BY/C-09/09. Authors would
also like to thank Dr. Murilo Cabral (University of Sao
Paulo) for useful discussions and help with the AutoDock
program.
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Nanomolar Detection of Glutamate