12 lactato_NTC_SNB

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Sensors and Actuators B 124 (2007) 269–276
Amperometric biosensor for lactate based on lactate dehydrogenase and
Meldola Blue coimmobilized on multi-wall carbon-nanotube
Arnaldo C. Pereira a,∗, Marina R. Aguiar b, Alexandre Kisner c,
Denise V. Macedo a, Lauro T. Kubota c
a Instituto de Biologia, Universidade Estadual de Campinas-UNICAMP, P.O. Box 6109, CEP 13083-970 Campinas/SP, Brazil
b Centro de Componentes Semicondutores, Universidade Estadual de Campinas-UNICAMP, P.O. Box 6061, CEP 13083-870 Campinas/SP, Brazil
c Instituto de Quı́mica, Universidade Estadual de Campinas-UNICAMP, P.O. Box 6154, CEP 13084-970 Campinas/SP, Brazil
Received 13 September 2006; received in revised form 14 December 2006; accepted 18 December 2006
Available online 28 December 2006
bstract
In this work, multi-wall carbon-nanotube (MWCT) is evaluated as transducer, stabilizer and immobilization matrix for the construction of amper-
metric biosensor based on lactate dehydrogenase (LDH) and Meldola’s Blue (MB). The amperometric response was based on the electrocatalytical
roperties of MB to oxidize NADH, which was generated in the enzymatic reaction of lactate with NAD+ under catalysis of LDH. It is shown
hat the employed materials are promising as electrochemical mediators and enzyme stabilizers. The enzyme was immobilized onto the MWCT
dsorbed with MB by cross-linking with glutaraldehyde. The dependence on the biosensor response for lactate was investigated in terms of pH,
upporting electrolyte, LDH and NAD+ amounts and applied potential. The amperometric response for lactate using this biosensor showed excellent
ensitivity (3.46 �A cm−2 mmol L−1), operational stability (around 96.5% of the activity was maintained after 300 determinations) and wide linear
esponse range (0.10–10 mmol L−1). These favorable characteristics allowed its application for direct measurements of lactate in blood samples.
ood agreement was found between the results obtained by the developed biosensor and other well established analytical method, indicating that
t should be highly selective for the lactate. Moreover, the biosensor also presented excellent stability in operational conditions.
2007 Elsevier B.V. All rights reserved.
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eywords: Carbon nanotube paste electrode; Enzyme biosensor; Lactate dehyd
. Introduction
Carbon nanotubes (CNT) have been intensely investigated
ince their discovery. This considerable interest reflects the
nique behavior of CNT, including their remarkable electrical,
hemical, mechanical and structural properties. CNT can
isplay metallic, semiconducting and superconducting electron
ransport, posses a hollow core suitable for storing guest
olecules and have the largest elastic modulus of any known
aterial [1–3].
CNT can be divided into single-wall carbon-nanotubes
SWCT) and multi-wall carbon-nanotubes (MWCT). SWCT
ossess a cylindrical nano-structure formed by rolling up a
ingle graphite sheet into a tube. MWCT comprise of several
∗ Corresponding author. Tel.: +55 19 3788 6146; fax: +55 19 3788 6129.
E-mail address: arnaldocsp@yahoo.com.br (A.C. Pereira).
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[
925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
oi:10.1016/j.snb.2006.12.042
ase; Meldola’s Blue; Lactate determination
ayers of graphene cylinders that are concentrically nested like
ings of a tree trunk. The unique properties of carbon nanotubes
ake them extremely attractive for the task of chemical sensors,
articularly for electrochemical detection [4–6]. Such potential
pplications would greatly benefit from the ability of carbon nan-
tubes to promote the electron-transfer reactions of important
iomolecules, including cytochrome c, NADH, catecholamine,
eurotransmitters, enzymes and ascorbic acid [7–9].
CNT paste enzyme electrodes were prepared by mixing
NT with mineral oil. Such composite electrode combines
he ability of carbon nanotubes to promote electron-transfer
eactions with the attractive advantages of paste electrode
aterials. These materials allow easy enzyme immobilization,
eproducible electrochemical behavior and useful physical char-
cteristics [10–12].
Measurement of lactate using biosensors is of great impor-
ance for the clinical analysis as well as for food analysis
13–18].
mailto:arnaldocsp@yahoo.com.br
dx.doi.org/10.1016/j.snb.2006.12.042
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70 A.C. Pereira et al. / Sensors an
Many methods have been reported for lactate determi-
ation, such as chromatographic and spectrometric analysis
19–22]. However, these methods are relatively expensive, time
onsuming, complex to perform and require laborious sam-
le pre-treatment. Thus, there is an increasing demand for
nexpensive, rapid and reliable methods for lactate determina-
ion. Electrochemical techniques, specially employing sensitive
mperometric biosensors, are particularly suited to this kind
f analysis [23–29]. The enzymes normally used in the devel-
pment of amperometric biosensors for lactate detection are
actate oxidase (LOD) and lactate dehydrogenase (LDH). In
he first case, O2 consumption or H2O2 production is moni-
ored. In an LDH-based biosensor, the enzyme catalyzes the
xidation of lactate to pyruvate in the presence of nicotinamide
denine dinucleotide (NAD+) and the reduced NADH can be
mperometrically detected.
This later approach has some important characteristics for
actate monitoring in real samples, such as: it is not oxygen
ependent and is more selective for lactate. However, the LDH-
ased biosensor has some drawbacks, including the fact that
hey are unstable [30] and the electrochemical oxidation of
ADH occurs, generally, at high over potentials [31,32]. These
igh potentials can allow interference from other electroac-
ive compounds usually present in real samples. Moreover, the
ntermediates produced during the oxidation reaction can lead
o electrode fouling [33]. Therefore, great attention has been
ocused on decreasing the over potential; using various inorganic
nd organic mediators on the electrodes for the electrochemical
xidation of NADH [34–37].
Thus, in order to provide an effective way for overcoming
he drawbacks of LDH-based biosensors, this paper describes
he development of an amperometric biosensor for lactate. This
evice is comprised of an amperometric biosensor for lactate
ased on the coimmobilization of lactate dehydrogenase and
eldola’s Blue (MB) on multi-wall carbon-nanotube through
he cross-linking with glutaraldehyde and agglutination with
ineral oil.
. Experimental
.1. Reagents
Lactate dehydrogenase (EC 1.1.1.27) 876 U mg−1 protein
10 mg protein mL−1) purchased from Sigma. Glutaraldehyde
as purchased from Fluka (Buchs, Switzerland). Multi-wall
arbon-nanotube (99%) was purchased from CNT Co. Ltd.,
ncheon, Korea. Meldola’s Blue was purchased from Aldrich
Milwaukee, USA, ≈99%). Graphite powder 99.9% from BDH
Poole, United Kingdom), bovine serum albumin (BSA) and
ineral oil (Sigma, St. Louis, MO, USA) were used to pre-
are the carbon-nanotube electrode. All other used reagents
ere of analytical grade. During the application of the pro-
osed biosensor employing biological samples, a commercial kit
upplied by Roche was used as reference method (spectrophoto-
etric), which possesses catalog number 1822837 found in the
ite (http://www.roche-diagnostica.com.br).
2
a
uators B 124 (2007) 269–276
.2. Mediator adsorption on multi-wall carbon-nanotubes
An aqueous solution of MB in a concentration of 5 ×
0−4 mol L−1 was used for the adsorption procedure. In 20 mL
f mediator solution, 0.1 g of MWCT was added and the mixture
as shaken for 2 h. The resulting solid was filtered and washed
5 times with deionized water, and then it was dried at 50 ◦C for
h. This material will be hereafter designated as MWCT-MB.
he quantity of immobilizedmediators was determined by ele-
ental analysis, using a Perkin-Elmer-2400 elemental analyzer.
.3. Preparation of the biosensor using multi-wall
arbon-nanotube
The enzyme was immobilized directly on MWCT by the
ross-linking method using glutaraldehyde 5% (v/v). For an
mount of 10 mg of MWCT-MB, 25 �L of LDH solution
219 U), 200 �g of NAD+, 5 �L of glutaraldehyde and 1 mg of
SA were added. This mixture was homogenized and dried in
refrigerator for 15 h.
The biosensor was prepared by mixing 10 mg of this
DH/MWCT-MB modified with 3 mg of graphite powder. After
hat, 30 �L of mineral oil was added and mixed until obtaining
homogeneous paste. This paste was placed into the cavity of a
lass tube with 4 mm internal diameter and 1 mm depth, obtain-
ng the modified nanotube paste electrode LDH/MWCT-MB.
ence, the developed biosensor contains all necessary reagents
mmobilized into the nanotube paste.
All the response obtained with the proposed biosensor were
iven in terms of current density. Thus, the geometric area of
he work electrode (biosensor) was determined as (A = πr2) and
t presented the value of 0.12 cm2.
.4. Electrochemical characterization and optimization of
he biosensor
The electrochemical measurements were performed, using a
otentiostat PGSTAT 30 model (Eco Chemie), interfaced with
personal computer for data acquisition and potential control.
ll electrochemical experiments were carried out in a conven-
ional three-electrode cell under room temperature, with a SCE
s the reference, a platinum wire as the counter electrode and
he modified carbon nanotube as the working electrode.
.5. Physical characterization of MWCT and
DH/MWCT-MB
Micrographic images were carried out using a LV-SEM
JEOL JSM 6360LV) equipped with an EDS (NORAN Sys-
em SIX Model 300). Fourier transform (FT) IR spectra were
easured with a Nicolet Magna-IR 550 spectrometer using a
Br pellet.
.6. Blood samples
The blood samples were taken from an athlete’s vein before
nd after the accomplishment of physical exercise with short
http://www.roche-diagnostica.com.br/
A.C. Pereira et al. / Sensors and Actuators B 124 (2007) 269–276 271
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(—) are shown in Fig. 2. As it can be clearly seem the spectrum
for the unmodified MWCT exhibits a significant difference in
comparison to the spectrum obtained with the modified MWCT.
The most outstanding features in this later are the three bands
ig. 1. LV-SEM image of MWCT (A) and LDH/MWCT-MB (B) by a factor of
ottom of (B) corresponds to a CNT containing LDH. (—) Means to 100 nm.
uration and high intensity. With the practical of physical exer-
ise, the lactate level increases quickly, however, simultaneously
he glicolysis in cells of the blood samples can increase the level
f the lactate. In this way, in order to avoid the interference
rom glicolysis, centrifuge tubes containing heparin previously
dsorbed on the walls, were used to separate the plasma of the
ells in a maximum range of 15 min after the sample’s collect.
his procedure is acceptable since the total blood is conserved
n ice.
The lactate determination with the proposed biosensor was
ompared with a spectrophotometric method. For this last, stan-
ard solutions of lactate in four different concentrations were
repared, where 9 �L of these were diluted in 900 �L of solu-
ion supplied by the Kit purchased from Roche and absorbance
ere measured (λ = 550 nm) in triplicate. In this way, a calibra-
ion curve for lactate determination in the plasma samples was
btained. The measurement with plasma samples employed the
ame procedure.
For the lactate determination in plasma using the proposed
iosensor, 500 �L of sample was diluted in 4.5 mL of phos-
hate buffer solution at pH 7.5, and following through the
reviously obtained calibration curve, it was determinated the
actate concentration in the plasma in two studied conditions.
he measurements were carried out in triplicate.
. Results and discussion
The MWCT used here are reported to have cylindrical geom-
try with a diameter of 10–40 nm and length of 5–20 �m.
The adsorption of MB on MWCT was carried out to
btain a maximum adsorption, and the amount adsorbed was
2 �mol g−1, which is higher than those obtained using other
dsorbent matrices [38,39].
The use of CNT as matrix for immobilization exhibits advan-
ages for chemically modified electrodes (CME) mainly in the
iversity of preparation method for sensors and biosensors. As
he CNT matrices are effective in the immobilization process
s transducer material, it is possible to use them together in the
omposite production, as carbon paste. The coupling between
he biocatalytic material and the electrode surface can be pro-
oted through the interaction between the functional groups of
he materials and the enzyme through the terminal amino acids.
F
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0. Inset the correspondent to EDS analysis. (—) Means to 2.5 �m. Inset at right
.1. Physical characterization of MWCT and
DH/MWCT-MB
The LV-SEM images of MWCT (A) and LDH/MWCT-MB
B) are depicted in Fig. 1. The comparison between both images
hows a significant difference in the morphology of materials.
n Fig. 1B, it is possible to observe an increase in the diameter
f CNT with the adsorbed enzyme. Moreover, through the EDS
nalysis (inset top right), it is possible to observe a difference
n the chemical composition of these materials. The amount of
ulfur presented by LDH/MWCT-MB material is much higher
han that presented by bare MWCT. This feature is an indicative
f the enzyme presence on the MWCT material. Moreover, FT-
R analysis also presented significant differences supporting the
btained data here.
The FTIR spectra of unmodified (· · ·) and modified MWCT
ig. 2. FTIR spectra of MWCT (· · ·) and LDH/MWCT-MB (—) in the KBr
ellet.
272 A.C. Pereira et al. / Sensors and Actuators B 124 (2007) 269–276
Fig. 3. Effect of MWCT electrode composition on the biosensor response for
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Table 1
Biosensor response as a function of the solution pH, obtained in 0.1 mol L−1
phosphate buffer solution, with 5 × 10−4 mol L−1 of lactate at an applied poten-
tial of 0 V vs. SCE
pH �j (�A cm−2)
5.5 0.21 ± 0.04
6.0 0.45 ± 0.04
6.5 1.17 ± 0.06
7.0 1.46 ± 0.06
7.5 1.46 ± 0.06
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The amperometric response was also investigated as a func-
tion of the amount of LDH in the carbon nanotube paste electrode
(data not shown). Five nanotubes paste electrode were prepared
with different amount of LDH in the range from 0.87 up to
Table 2
Amperometric response obtained for LDH/MWCT-MB lactate biosensor at dif-
ferent applied potentials
E (mV) vs. SCE �j (�A cm−2)
−300 0.06 ± 0.01
−200 0.53 ± 0.03
−100 1.28 ± 0.05
−50 1.43 ± 0.05
0 1.47 ± 0.06
actate obtained for LDH/graphite (A), LDH/MWCT (B) and LDH/MWCT-MB
C). Applied potential of 0.0 V vs. SCE, 0.1 mol L−1 phosphate buffer solution,
H 7.5.
n 1400, 1109 and 615 cm−1, which could be attributed to the
romatic groups (–Cs C–), C–C or C–N bending and C–H
ut-of-plane bending of aromatic groups of enzyme structure
40], respectively. Moreover, the large band in 3422 cm−1 (–OH
tretch on MWCT surface) in the unmodified MWCT spectra
hift to lower frequency in the modified one. This slight shift
ay be associated with the hydrogen bonding between the –OH
roups on MWCT surface and the hydrophilic groups (–NH–,
O) of the enzyme structure.
.2. Lactate biosensor
The electrocatalytic oxidation of NADH in the presence of
B has been previously reported [31,33]. The present results
how that the MWCT-MB paste electrode can also catalyze
he oxidation of NADH that is generated from the reaction of
AD+ and lactate catalyzed by LDH. Since NAD+ and media-
or concentration are constant, the increase in the electrocatalytic
urrent depends only on the lactate concentration. This charac-
eristic was used as the base of the biosensor development for
actatedetermination. Therefore, the complete operation of the
iosensor, include both enzymatic and electrochemical reaction.
.3. Optimization of the biosensor compounds
Fig. 3 shows the effect of MWCT electrode composition on
he biosensor response for lactate, the amperometric measure-
ents were carried out at 0.1 mol L−1 phosphate buffer, pH 7.5.
he analytical curves obtained using LDH/graphite (Fig. 3A),
DH/MWCT (Fig. 3B) showed low response at an applied
otential of 0.0 mV versus SCE. Between these two curves an
mportant increase in the response was observed and attributed to
he ability of the MWCT matrix to act as transducer for amper-
metric measurements. This is due to high conductivity, high
L
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.0 1.45 ± 0.06
.5 1.23 ± 0.05
urface area facilitating the electron transfer. However, for the
DH/MWCT-MB (Fig. 3C) a significant catalytic current was
bserved, which provided an excellent sensitivity for lactate
2.7 �A cm−2 mmol L−1). These data confirm the efficiency of
oth: MB as an electron mediator on the electrode surface and
f carbon nanotube as enzyme immobilization matrix.
The influence of the solution pH on the biosensor response for
actate was investigated in the pH range between 5.5 and 8.5. As
s shown in Table 1, the current density is enhanced by increasing
he solution pH from 6.0 to 7.0, reaching a maximum value for
H between 7.0 and 8.0. This pH range reflected the optimum
onditions for both the enzymatic and mediated electrochemical
eactions in the carbon nanotube electrode. Thus, the solution
H used in the experiments was 7.5.
The dependence of the applied potential on current of the
DH/MWCT-MB biosensor was tested in the range of −300
o 100 mV versus SCE (Table 2). The current increased greatly
hen the applied potential shifted toward more positive value
ith an excellent signal-to-noise ratio at 0.0 V versus SCE. In
his way, 0 mV versus SCE was chosen as the optimum working
otential since such potential achieves a high current and it is less
usceptible to the interference of easily oxidizable and reducible
pecies. Thus, this biosensor is less vulnerable to the interfer-
nce from several compounds commonly present in real complex
amples. In addition, at these potentials, low noise levels and low
ackground currents were observed.
50 1.47 ± 0.06
100 1.47 ± 0.05
actate concentration of 5.0 × 10−4 prepared in 0.1 mol L−1 phosphate buffer,
H 7.5.
A.C. Pereira et al. / Sensors and Actuators B 124 (2007) 269–276 273
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Fig. 5. Analytical curves obtained using the biosensor prepared with BSA
and LDH immobilized by physical adsorption (A) and by cross-linking with
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modified electrodes showed that NADH and mediator can form
a stable charge-transfer complex with a low tendency to disso-
ciation.
ig. 4. Amperometric response to successive addition of 0.50 mM lactate
btained for the developed biosensor. Applied potential is 0.0 V vs. SCE in
.1 mol L−1 phosphate buffer solution, pH 7.5. [LDH] = 4.4 U mg−1.
7.5 U of enzyme per mg of nanotube, while the amounts of other
omponents were kept constant. The sensitivity of the modified
lectrode was dependent on the amount of enzyme incorporated
n the nanotube. An increase in the sensitivity was observed up to
.4 U mg−1 of paste, while for higher loads no increase was ver-
fied. Therefore, all further experiments were carried out using
.4 U mg−1 of paste. Fig. 4 shows the amperometric response
btained for the developed biosensor employing 4.4 U of LDH
er mg of nanotube.
When an enzyme is immobilized on solid supports with large
ctive surface, the surface groups on the support can induce
onformational changes on the enzyme and can lead to lose
he activity. It is well known that covalent attachment of an
nzyme results in a more stable immobilization compared to
hose obtained by physical adsorption. In order to improve the
tabilization and LDH activity, the effect of the immobiliza-
ion method was investigated. Fig. 5 shows the response of the
iosensor prepared with bovine serum albumin (BSA) incorpo-
ated into nanotube and LDH without glutaraldehyde (Fig. 5A)
nd with glutaraldehyde (Fig. 5B). These results show that the
mmobilization procedure with glutaraldehyde/BSA gives better
ensitivity and a wider linear response range than that observed
or physical adsorption.
The NAD+ coenzyme also plays an essential role in the
iosensor mechanism. Previous studies [18,41] showed that
hen NAD+ is incorporated into carbon paste the slopes of
he lactate calibration plots (sensitivity) are higher than those
btained using NAD+ free in solution.
Thus, the effect of the amount of NAD+ immobilized on the
roposed biosensor response was also evaluated. A plot of the
iosensor sensitivity as a function of the NAD+ percentage in
he carbon paste is shown in Fig. 6. As can be observed, the
esponse increases sharply with increasing NAD+ percentages
ntil 0.40% (m/m); for higher NAD+ percentages, the sensitivity
ecreases. Based on these results, 0.40% of NAD+ (m/m) or
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lutaraldehyde (B). Experiment carried out in 0.1 mol L−1 phosphate buffer
olution, pH 7.5 and potential step to 0.0 mV vs. SCE.
00 �g of NAD+, was employed in the development of all the
ubsequent biosensors. Although NAD+ is highly soluble, no
ecrease in the response was observed.
This behavior suggests that during the glutaraldehyde reac-
ion for enzyme immobilization, the NAD+ was efficiently
ccluded into the polymeric net formed with BSA, LDH and
lutaraldehyde, avoiding the NAD+ leaches out from the car-
on nanotube paste. In addition, previous studies [31,33] using
ig. 6. LDH/MWCT-MB based biosensor sensitivity as a function of the NAD+
ass incorporated into the carbon paste. Potential step to 0.0 V vs. SCE,
.1 mol L−1 phosphate buffer solution (pH 7.0). Sensitivity obtained by full
alibration curves.
274 A.C. Pereira et al. / Sensors and Actuators B 124 (2007) 269–276
Table 3
Determination of lactate in plasma samples
Sample Reference method (mmol L−1) Biosensor (mmol L−1) t-Student theoreticala t-Student calculated
#1 2.52 ± 0.04 2.44 ± 0.06 2.77 1.93
# 12 2.77 1.92
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2 5.56 ± 0.08 5.72 ± 0.
a t-Student test for 95% of confidence level.
The decrease in the sensitivity observed in Fig. 6 can be
xplained due to a competition for the active site of the enzyme
y the NAD+ and substrate. Thus, increasing the NAD+ amount
an difficult to access of the active site of the enzyme by the
ubstrate.
.4. Analytical curve for lactate
The LDH modified nanotube paste based biosensor showed
n excellent sensitivity for lactate in a wide linear response
ange, obtaining a strictly linear calibration curve from 0.1 to
0 mmol L−1 of lactate in 0.1 mol L−1 phosphate buffer at pH
.5. This analytical curve was described by the equation: �j
�A cm−2) = 0.06 (±0.02) + 3.46 (±0.01) [lactate] (mmol L−1)
ith a correlation coefficient of 0.9995 for n = 21. The developed
iosensor presented an excellent operational range and good
ensitivity. Detection limits around 7.5 × 10−6 mol L−1 could
e estimated considering 3σB/slope. This modified electrode
resented excellent repeatability, with a relative S.D. of 2.3% for
series of seven successive measurements of 5 × 10−4 mol L−1
actate solution. This biosensor was very stable allowing
etermination of 300 samples without significant change or
ore than 8 h in continuous use (around 96.5% of the activity
as maintained after 300 determinations). The linear response
ange, detection limit and, especially, the stability presented
y the proposed biosensor are quite superior to the most of the
mperometric biosensors for lactate described in the literature
42–51].
.5. Lactate determination in blood
The favorable characteristics presented by the proposed
iosensor allowed its application for the direct determination of
actate in real samples. Hence, theperformance of the biosen-
or was tested by applying it for the determination of lactate in
lood samples, before (sample 1) and after (sample 2) a physical
xercise with short duration and high intensity.
Table 3 summarizes the concentrations found by the biosen-
or and reference method (commercial Kit). As can be observed,
he data showed an excellent correlation between the results
btained with the biosensor and with reference method (spec-
rophotometric). Applying the t-Student test, it was possible to
erify that the averages obtained by the both methods are statis-
ically equal in the confidence level of 95%. Previously, F-test
as applied to show that the precisions of the both methods are
he same. This performance presented by the developed biosen-
or indicates that it should be highly selective for the lactate.
oreover, the biosensor also presented excellent stability in
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ig. 7. Operational stability obtained for the biosensor in plasma sample; poten-
ial step to 0.0 V vs. SCE, 0.1 mol L−1 phosphate buffer solution (pH 7.5).
perational conditions (measured in biological sample). As can
e observed in Fig. 7, around 94% of the initial response was
aintained after 20 determinations.
On the other hand, the proposed amperometric biosensor
resents some advantages in relation to the standard method,
uch as analysis time and the chemicals required preparing
iosensor is lower than those used for standard method. More-
ver, the biosensor preparation in one step associated to the
ossibility of the coenzyme and enzyme regeneration makes
ower the cost per analysis.
. Conclusion
The results demonstrated that the multi-wall carbon-nanotube
aterial is an efficient support for MB adsorption providing
n excellent environment to NADH oxidation as well for LDH
mmobilization. Therefore, this material was very useful for a
imple and effective way to develop reagentless amperomet-
ic biosensors highly sensitive for lactate determination. The
xperiments described above illustrate the ability to employ
his biosensor for lactate detection in real samples, without
dding any reagent. The sensitivity (3.46 �A cm−2 mmol L−1),
etection limit (7.5 × 10−6 mol L−1) and linear response range
from 0.1 to 10 mmol L−1) make this biosensor an excellent
evice for lactate determination. Moreover, the proposed LDH-
ased biosensor exhibited an excellent operational and storage
tability.
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A.C. Pereira et al. / Sensors an
cknowledgements
The authors thank FAPESP for the financial support. A.C.P.
s indebted to FAPESP for the fellowships.
eferences
[1] Y.J. Kim, T.S. Shin, H.D. Choi, J.H. Kwon, Y.-C. Chung, H.G. Yoon,
Electrical conductivity of chemically modified multiwalled carbon nan-
otube/epoxy composites, Carbon 43 (2005) 23–30.
[2] H. Miyagawa, M. Misra, A.K. Mohanty, Mechanical properties of carbon
nanotubes and their polymer nanocomposites, J. Nanosci. Nanotechnol. 5
(2005) 1593–1615.
[3] S. Sotiropoulou, V. Gavalas, V. Vamvakaki, N.A. Chaniotakis, Novel
carbon materials in biosensor systems, Biosens. Bioelectron. 18 (2003)
211–215.
[4] Y.H. Lin, W. Yantasee, J. Wang, Carbon nanotubes (CNTs) for the devel-
opment of electrochemical biosensors, Front. Biosci. 10 (2005) 492–505.
[5] A. Merkoci, M. Pumera, X. Lopis, B. Perez, M. Del Valle, S. Alegret, New
materials for electrochemical sensing VI: carbon nanotubes, TRAC-Trends
Anal. Chem. 24 (2005) 826–838.
[6] J.J. Gooding, Nanostructuring electrodes with carbon nanotubes: a review
on electrochemistry and applications for sensing, Electrochim. Acta 50
(2005) 3049–3060.
[7] J. Wang, Carbon-nanotube based electrochemical biosensors: a review,
Electroanalysis 17 (2005) 7–14.
[8] M.K. Wang, Y. Shen, Y. Liu, T. Wang, F. Zhao, B.F. Liu, S.J. Dong, Direct
electrochemistry of microperoxidase 11 using carbon nanotube modified
electrodes, J. Electroanal. Chem. 578 (2005) 121–127.
[9] M.D. Rubianes, G.A. Rivas, Enzymatic biosensors based on carbon nan-
otubes paste electrodes, Electroanalysis 17 (2005) 73–78.
10] Q. Zhao, L. Guan, G. Zhennan, Z. Qiankun, Determination of phenolic
compounds based on the tyrosinase-single walled carbon nanotubes sensor,
Electroanalysis 17 (2005) 85–88.
11] K. Yamamoto, G. Shi, T. Zhou, F. Xu, J. Xu, T. Kato, J.-Y. Jin, L. Jin,
Study of carbon nanotubes—HRP modified electrode and its application
for novel on-line biosensors, Analyst 128 (2003) 249–254.
12] J.Z. Xu, J.J. Zhu, Q. Wu, Z. Hu, H.Y. Chen, An amperometric biosensor
based on the coimmobilization of horseradish peroxidase and methylene
blue on a carbon nanotubes modified electrode, Electroanalysis 15 (2003)
219–224.
13] K. Davranche, M. Audiffren, Facilitating effects of exercise on information
processing, J. Sport Sci. 22 (2004) 419–428.
14] S. Maldonado-Martin, I. Mujika, S. Padilla, Physiological variables to use
in the gender comparison in highly trained runners, J. Sport Med. Phys.
Fit. 44 (2004) 8–14.
15] A. McAinch, M.A. Febbraio, J.M. Parkin, S.A. Zhao, K. Tangalakis,
L. Stojanovska, M.F. Carey, Effect of active versus passive recovery on
metabolism and performance during subsequent exercise, Int. J. Sport Nutr.
Exe. 14 (2004) 185.
16] E. Bonanni, L. Pasquali, M.L. Manca, M. Maestri, C. Prontera, M. Fab-
brini, S. Berrettini, G. Zucchelli, G. Siciliano, L. Murri, Lactate production
and catecholamine profile during aerobic exercise in normotensive OSAS
patients, Sleep Med. 5 (2004) 137–145.
17] N. Sato, H. Okuma, Amperometric simultaneous sensing system for d-
glucose and l-lactate based on enzyme-modified bilayer electrodes, Anal.
Chim. Acta 565 (2006) 250–254.
18] A.C. Pereira, D.V. Macedo, A.S. Santos, L.T. Kubota, Amperometric
biosensor for lactate based on Meldola’s blue adsorbed on silica gel mod-
ified with niobium oxide, Electroanalysis 18 (2006) 1208–1214.
19] H. Bariskaner, M.E. Ustun, A. Ak, A. Yosunkaya, N. Dogan, M. Gurbilek,
Effects of deferoxamine on tissue lactate and malondialdehyde levels in
cerebral ischemia, Method Find Exp. Clin. Pharmac. 25 (2003) 371–376.
20] B.A. Posner, L.Y. Li, R. Bethell, T. Tsuji, S.J. Benkovic, Engineering
specificity for folate into dihydrofolate reductase from Escherichia coli,
Biochemistry 35 (1996) 1653–1663.
[
uators B 124 (2007) 269–276 275
21] L. Fernandes, A.M. Relva, M.D.R.G. da Silva, A.M.C. Freitas, Different
multidimensional chromatographic approaches applied to the study of wine
malolactic fermentation, J. Chromatogr. A 995 (2003) 161–169.
22] R.W. Wulkan, R. Verwers, M. Neele, M.J. Mantel, Measurement of
pyruvate in blood by high-performance liquid chromatography with flu-
orescence detection, Annals Clin. Biochem. 38 (2001) 554–558.
23] F.L. Qu, M.H. Yang, J.H. Jiang, G.L. Shen, R.Q. Yu, Amperometric biosen-
sor for choline based on layer-by-layer assembled functionalized carbon
nanotube and polyaniline multilayer film, Anal. Biochem. 344 (2005)
108–114.
24] L.Y. Jiang, C.X. Liu, H.Q. Li, X.B. Luo, Y.R. Wu, X.X. Cai, Performance
of an amperometric biosensor for the determination of hemoglobin, J.
Nanosci. Nanotechnol. 5 (2005) 1301–1304.
25] N.G. Patel, S. Meier, K. Cammann, G.C. Cheminitius, Screen-printed
biosensors using different alcohol oxidases, Sens. Actuators, B Chem. 75
(2001) 101–110.
26] H. Gulce, A. Gulce, M. Kavanoz, H. Coskun, A. Yildiz, A new ampero-
metric enzyme electrode for alcohol determination, Biosens. Bioelectron.
17 (2002) 517–521.
27] G. Li, N.Z. Ma, Y. Wang, A new handheld biosensor for monitoring blood
ketones, Sens. Actuators, B Chem. 109 (2005) 285–290.
28] A. Salimi, A. Noorbakhsh, M. Ghadermarz, Direct electrochemistry and
electrocatalytic activity of catalase incorporated onto multiwall carbon
nanotubes-modified glassy carbon electrode, Anal. Biochem. 344 (2005)
16–24.
29] R.C.H. Kwan, H.F. Leung, P.Y.T. Hon, H.C.F. Cheung, K. Hirota, R.
Renneberg, Amperometric biosensor for determining human salivary phos-
phate, Anal. Biochem. 343 (2005)263–267.
30] M.J.F. Rebelo, D. Compaguone, G.G. Guilbault, Alcohol electrodes in
beverage measurements, Anal. Lett. 27 (1994) 3027–3037.
31] A.S. Santos, L. Gorton, L.T. Kubota, Nile blue adsorbed onto silica gel
modified with niobium oxide for electrocatalytic oxidation of NADH,
Electrochim. Acta 47 (2002) 3351–3360.
32] L. Gorton, E. Dominguez, Encyclopedia of electrochemistry, in: A.J. Bard,
M. Stratmann (Eds.), Bioelectrochemistry, vol. 9, Wiley-VCH, 2002, p. 67.
33] A.S. Santos, L. Gorton, L.T. Kubota, Electrocatalytic NADH oxidation
using an electrode based on Meldola blue immobilized on silica coated
with niobium oxide, Electroanalysis 14 (2002) 805–812.
34] P.N. Bartlett, in: R.P. Buck (Ed.), Biosensor Technology Fundamentals and
Applications, Dekker, New York.
35] L. Gorton, E. Csoregi, E. Domı́nguez, J. Emneus, G. Jönsson-Pettersson, G.
Marko-Varga, B. Persson, Selective detection in flow-analysis based on the
combination of immobilized enzymes and chemically modified electrodes,
Anal. Chim. Acta 250 (1991) 203–248.
36] P.C. Pandey, S. Upadhyay, H.C. Pathak, C.M.D. Pandey, Sensitivity,
selectivity and reproducibility of some mediated electrochemical biosen-
sors/sensors, Anal. Lett. 31 (1998) 2327–2348.
37] P. Wang, Y. Yuan, G.Y. Zhu, Carbon ceramic electrodes modified
with sub-micron particles of new methylene blue (NMB) intercalated
alpha-zirconium phosphate, J. Electroanal. Chem. 519 (2002) 130–
136.
38] A.S. Santos, R.S. Freire, L.T. Kubota, Highly stable amperometric biosen-
sor for ethanol based on Meldola’s blue adsorbed on silica gel modified
with niobium oxide, J. Electroanal. Chem. 547 (2003) 135–142.
39] F.A. Pavan, E.S. Ribeiro, Y. Gushikem, Congo red immobilized on a sil-
ica/aniline xerogel: preparation and application as an amperometric sensor
for ascorbic acid, Electroanalysis 17 (2005) 625–629.
40] C. Sass, M. Briand, S. Benslimane, M. Renaud, Y. Briand, Characterization
of rabit lactate dehydrogenase-M and lactate dehydrogenase-H cDNAs, J.
Biol. Chem. 264 (1989) 4076–4081.
41] A.S. Santos, A.C. Pereira, N. Duran, L.T. Kubota, Amperometric biosensor
for ethanol based on co-immobilization of alcohol dehydrogenase and Mel-
dola’s Blue on multi-wall carbon nanotube, Electrochim. Acta 52 (2006)
215–220.
42] K. Matsumoto, H. Matsubara, M. Hamada, H. Ukeda, Y. Osajima, Simulta-
neous determination of glucose, ethanol and lactate in alcoholic beverages
and serum by amperometric flow-injection analysis with immobilized
enzyme reactors, J. Biotechnol. 14 (1990) 115–126.
2 d Act
[
[
[
[
[
[
[
[
[
B
A
C
H
s
M
S
A
D
S
H
Lauro Tatsuo Kubota has a PhD in inorganic chemistry at UNICAMP in 1994.
76 A.C. Pereira et al. / Sensors an
43] E. Dempsey, J. Wang, M.R. Smyth, Electropolymerized o-phenylenedia-
mine film as means of immobilising lactate oxidase for a l-lactate biosensor,
Talanta 40 (1993) 445–451.
44] W.A. Collier, D. Janssen, A.L. Hart, Measurement of soluble l-lactate
in dairy products using screen-printed sensors in batch mode, Biosens.
Bioelectron. 11 (1996) 1041–1049.
45] A.L. Hart, C. Matthews, W.A. Collier, Estimation of lactate in meat extracts
by screen-printed sensors, Anal. Chim. Acta 386 (1999) 7–12.
46] K. Kriz, L. Kraft, M. Krook, D. Kriz, Amperometric determination of l-
lactate based on entrapment of lactate oxidase on a transducer surface with
a semi-permeable membrane using a SIRE technology based biosensor.
Application: tomato paste and baby food, J. Agric. Food Chem. 50 (2002)
3419–3424.
47] S.C. Litescu, L. Rotariu, C. Bala, Immobilisation of lactate dehidrogenase
on electro-polymerised Meldola blue matrix, Revista de Chimie 56 (2005)
57–60.
48] R. Gajonyte, V. Melvydar, A. Malinauskas, Mediated amperometric biosen-
sors for lactic acid based on carbon paste electrodes modified with Baker’s
yeast Saccharomyces cerevisiae, Bioelectrochemistry 68 (2006) 191–196.
49] J. Weber, A. Kumar, A. Kumar, S. Bhansali, Novel lactate and pH biosensor
for skin and sweat analysis based on single walled carbon nanotubes, Sens.
Actuators, B Chem. 117 (2006) 308–313.
50] I. Rohm, M. Genrich, W. Collier, U. Bilitewski, Development of ultraviolet-
polymerizable enzyme pastes: bioprocess applications of screen-printed
l-lactate sensors, Analyst 121 (1996) 877–881.
H
h
o
uators B 124 (2007) 269–276
51] W.A. Collier, P. Lovejoy, A.L. Hart, Estimation of soluble l-lactate in dairy
products using screen-printed sensors in a flow injection analyser, Biosens.
Bioelectron. 13 (1998) 219–225.
iographies
rnaldo César Pereira has a PhD in analytical chemistry obtained at UNI-
AMP in 2003. He is currently a post doctorate student at UNICAMP, Brazil.
is research work has been mainly focused on the design of new electrochemical
ensors.
arina Rodrigues Aguiar ia a post doctorate student at Centro de Componentes
emicondutores, UNICAMP, Brazil.
lexandre Kisner is a MSc student at UNICAMP.
enise Vaz de Macedo has a PhD in biology obtained at UNICAMP in 1993.
he is currently an associate professor of biochemistry at UNICAMP, Brazil.
er research work has been focused in the biochemistry of physical activities.
e is currently a professor at UNICAMP, Campinas, Brazil. His research work
as been mainly focused on new electrochemical methods and also the design
f new electrochemical sensors.
	Amperometric biosensor for lactate based on lactate dehydrogenase and Meldola Blue coimmobilized on multi-wall carbon-nanotube
	Introduction
	Experimental
	Reagents
	Mediator adsorption on multi-wall carbon-nanotubes
	Preparation of the biosensor using multi-wall carbon-nanotube
	Electrochemical characterization and optimization of the biosensor
	Physical characterization of MWCT and LDH/MWCT-MB
	Blood samples
	Results and discussion
	Physical characterization of MWCT and LDH/MWCT-MB
	Lactate biosensor
	Optimization of the biosensor compounds
	Analytical curve for lactate
	Lactate determination in blood
	Conclusion
	Acknowledgements
	References