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Amperometric Biosensor for Lactate Based on Meldola�s Blue
Adsorbed on Silica Gel Modified with Niobium Oxide
Arnaldo C. Pereira,*a Denise V. Macedo,a Antonio S. Santos,b Lauro T. Kubotab
a Instituto de Biologia, Universidade Estadual de Campinas-UNICAMP, P. O. Box 6154 CEP 13084-862, Campinas/SP, Brazil
*e-mail: arnaldocsp@yahoo.com.br
b Instituto de Qu1mica, Universidade de Campinas-UNICAMP, P. O. Box 6109 CEP 13083-970, Campinas/SP, Brazil
Received: January 26, 2006
Accepted: March 31, 2006
Abstract
A reagentless amperometric biosensor sensitive to lactate was developed. This sensor comprises a carbon paste
electrode modified with lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NADþ) cofactor and
Meldola>s blue (MB) adsorbed on silica gel coated with niobium oxide. The amperometric response was based on the
electrocatalytic properties of MB to oxidize NADH, which was generated in the enzymatic reaction of lactate with
NADþ under catalysis of LDH. The dependence on the biosensor response was investigated in terms of pH,
supporting electrolyte, ionic strength, LDH and NADþ amounts and applied potential. The biosensor showed an
excellent operational stability (95% of the activity was maintained after 250 determinations) and storage stability
(allowing measurements for over than 2.5 months, when stored in a refrigerator). The proposed biosensor also
presented good sensitivity allowing lactate quantification at levels down to 6.5� 10�6 mol L�1. Moreover, the
biosensor showed a wide linear response range (from 0.1 to 14 mmol L�1 for lactate). These favorable characteristics
allowed its application for direct measurements of lactate in biological samples such as blood. The precision of the
data obtained by the proposed biosensor show reliable results for real complex matrices.
Keywords: Amperometric biosensor, Modified silica gel, NADþ, Lactate dehydrogenase, Lactate determination
DOI: 10.1002/elan.200603509
1. Introduction
Measurement of lactate using biosensors is of great impor-
tance for the clinical analysis as well as for food analysis [1 –
4]. For food chemistry, it is useful for evaluating freshness
and stability of milk, dairy products, fruits, vegetables,
sausages and wines. For clinical analysis, it is helpful for
monitoring respiratory insufficiency, shocks, heart failure
and metabolic disorder. It is also useful for detecting tissues
injury, thrombosis and physical condition of racing animals
and athletes.
Many methods have been reported for lactate determi-
nation, such as chromatographic and spectrometric analysis
[5 – 8]. However, these methods are relatively expensive,
time consuming, complex to perform and require laborious
sample pretreatment. Thus, there is an increasing demand
for inexpensive, rapid and reliable methods for lactate
determination. Electrochemical techniques, specially em-
ploying sensitive amperometric biosensors, are particularly
suited to this kind of analysis [9 – 17]. These devices have
many favorable analytical characteristics, such as selectivity,
sensitivity, portability, speed, low cost and potential for
miniaturization [18, 19]. The enzymes normally used in the
development of amperometric biosensors for lactate detec-
tion are lactate oxidase (LOD) and lactate dehydrogenase
(LDH). In the first case, O2 consumption or H2O2 produc-
tion is monitored. In an LDH-based biosensor, the enzyme
catalyzes the oxidation of lactate to pyruvate in the presence
of nicotinamide adenine dinucleotide (NADþ) and the
reduced NADH can be detected amperometrically, accord-
ing to the following reactions:
CH3CHOHCOO� þNADþ �!
LDH CH3COCOO� þ
NADHþHþ
NADH �!electrode NADþ þHþ þ 2e�
This last approach has some important characteristics for
the lactate monitoring in real samples, such as, it is not
oxygen dependent and more selective for lactate. However,
electrochemical biosensors based on dehydrogenases are
limited due to the difficulties involved in the electrochem-
ical detection or regeneration of the cofactors [20]. Both the
anodic oxidation NADH and cathodic reduction of NADþ
are highly chemically irreversible and need high over
potentials [21], frequently an overpotential higher than
1.0 V for the electrooxidation of NADH is necessary [22].
Moreover, side products may form during the direct
electrode processes, these species and NADþ can adsorb
on the electrode surface causing the electrode fouling [23],
especially for high concentrations of the cofactor. The
relative high potential necessary to electro oxidize NADH
may open up the sensing system to many interfering electro
active compounds [24] usually present in real samples. The
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use of electron transfer mediators immobilized on the
electrode surface to produce a chemically modified elec-
trode for this purpose minimizing these problems has been
widely studied in the last two decades [25, 26]. The recent
works involving the development of lactate sensor present-
ed in the literature, employ electron mediators very known
and studied such as Brilliant Cresyl [27], Ospoly(vivylpyr-
idine) [28] and neutral red-doped silica [29]. Besides these,
Ricci et al. [30] present in a review some works employing
Prussian Blue as electron mediator, in the development of
glucose, lactate, cholesterol andgalactose sensors.However,
the largest innovation in the development for lactate sensor
is presented by Rubianes et al. [31], in which the authors
used multi wall carbon nanotube (MWCNT) in the bio-
sensor preparation, where this material acts as electron
mediator.
However, the incorporation of species in the carbon
pastes still needs improvements, mainly in terms of stability
and reproducibility. Some works employing inorganic
materials such as silica gel and zeolites have been published
[32 – 34]. When adsorbed on these kinds of materials the
mediators show an improved electrochemical activity and/
or a shift of the redox potential of the mediator into a more
desirable potential range [35].
Silica gel has been employed as an inert support to graft
species in the preparation of modified electrodes [36, 37].
The versatility of thismaterial in immobilizingmany species,
while retaining its general properties such as rigidity,
porosity, particle size, high specific surface area and
chemical stability is very attractive for the development of
new sensors and biosensors [38]. In a previous study [39], it
was observed that the immobilization of Meldola>s blue
(MB) in silica gel modified with niobium oxide presented a
great potential to the development of new NADH sensors.
When adsorbed on silica gel modified with niobium oxide
the Meldola>s blue mediator showed an excellent stability
(due to a strong adsorption of the mediator on the modified
silica surface), improvement in the electrochemical activity
and a shift of the redox potential into amore intended range,
allowing an effective electro-oxidation of NADH at an
applied potential of 0.0 V vs. saturated calomel electrode
(SCE). Thus, in order to provide an effective way for
overcoming the drawbacks of LDH-based biosensors, this
paper describes the development of an amperometric
biosensor for lactate based on modified silica gel. The
main novelty of the design is the incorporation of NADþ to
the system. Optimization of operational conditions and
performance of this device are reported. Its practical
application for fast and reliable determination of lactate in
blood is also described.
2. Experimental
2.1. Reagents
Silica gel was purchased from Fluka.Meldola blue, niobium
pentachloride, graphite powder (99.9%),mineral oil, bovine
serumalbumin (BSA), lactate dehydrogenase (EC1.1.1.27),
295 U mg�1, glutaraldehyde, lactate and nicotinamide
adenine dinucleotide were chemicals of analytical grade
acquired from Aldrich or Sigma. During the application of
the proposed biosensor employing biological samples, a
commercial kit supplied by Roche was usedas reference
method (spectrophotometric), which possesses catalog
number 1822837 found in the site (www.roche-diagnostica.-
com.br).
2.2. Silica Modification and Characterization
The modification of silica gel surface with niobium oxide
and the adsorption of Meldola>s blue on the modified silica
were carried out as described by Santos et al. [39]. The
specific surface area of the silicawas determined by theBET
method, using aMicromeritics FlowSorb II 2300 (Norcross).
The amount of niobium grafted onto the silica gel was
determined by X-ray fluorescence using a spectrometer
Tracor 5000 model (Spectrace). The Meldola blue quantity
adsorbed on the silica surface was determined by elemental
analysis, using an Elemental Analyzer PE-2400 model
(Perkin Elmer).
2.3. Enzyme Immobilization and Modified Carbon Paste
Electrode Preparation
The enzyme was immobilized on graphite powder by the
cross-linking method using bovine serum albumin and
glutaraldehyde. For an amount of 20 mg of graphite powder,
1 mg of LDH dissolved in 80 mL of water, 200 mg of NADþ,
5 mL of glutaraldehyde 5% (v/v) and 1 mg of BSA were
added. This mixture was homogenized and dried in a
refrigerator for 15 hours.
The biosensor was preparedmixing 20 mg of this graphite
powdermodified with 20 mg ofMeldola>s Blue adsorbed on
silica gel modified with niobium oxide (SNMB). After that,
a small amount (30 mL) of mineral oil was added and mixed
until obtaining a homogeneous paste. This paste was placed
into the cavity of a glass tube with 4 mm internal diameter,
obtaining the modified carbon paste electrode (CPE).
Hence, the developed biosensor contains all necessary
reagents immobilized into the carbon paste.
All the response obtained with the proposed biosensor
were given in terms of current density. This way the
geometric area of the work electrode (biosensor) it was
determined as (A¼pr2) and it presented the value of
0.12 cm2.
2.4. Electrochemical Measurements
The electrochemical measurements were performed using a
potentiostat PGSTAT 30 model (PENSALAB), interfaced
with a personal computer for data acquisition and potential
control.All electrochemical experimentswere carriedout in
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a conventional three-electrode cell under room temper-
ature, with a saturated calomel electrode (SCE, Corning) as
the reference, a platinum wire as counter and the modified
carbon paste as working electrode.
2.5. Biological Samples
The blood samples were taken from an athlete>s vein before
and after the accomplishment of physical exercisewith short
duration and high intensity. With the practical of physical
exercise, the lactate level increases quickly, however,
simultaneously the glycolysis in cells of the blood samples
can increase the lactate level too. In this way, in order to
avoid the interference from glycolysis, centrifuge tubes
containing heparin previously adsorbed on the walls, were
used to separate the plasma of the cells in a maximum range
of 15 minutes after the sample>s collect. This procedure is
acceptable since the total blood is conserved in ice.
The lactate determination with the proposed biosensor
was compared with a spectrophotometric method. For this
last, standard solutions of lactate in 4 different concentra-
tions were prepared, where 9 mL of these were diluted in
900 mL of solution supplied by the Kit purchased from
Roche and absorbance measures (l¼ 550 nm) were ob-
tained in triplicate. This way, a calibration curve for lactate
determination in the plasma samples was obtained. The
measures with plasma samples employed the same proce-
dure.
For the determination of the lactate concentration in
plasma using the proposed biosensor, 500 mL of sample was
diluted in 4.5 mL of phosphate buffer solution pH 7.8, and
following through the calibration curve previously obtained,
it was determinated the lactate concentration in plasma in
the two studied conditions. Themeasureswere carried out in
triplicate.
3. Results and Discussion
3.1. Characteristics of the Material
The coating to disperse niobium oxide on the silica gel
surface allowed grafting niobium in a quantity of 1.5 mmol
g�1. This quantity is higher than those described in the
literature [40]. The equation that describes the niobium
grafting reactions can be represented as:
n(�SiOH)þNbCl5! (�SiO)nNbCl5�nþ nHCl
(�SiO)nNbCl5�nþ (5� n)H2O! (�SiO)nNb(OH)5�nþ
(5� n)HCl
The hydroxyl groups of the niobium are known to be acid.
The characterization of the material was published by
Denofre and co-workers [41], and it can be used to
immobilize basic substance like MB. The surface areas of
the silica before and after modification were 520 and 488 m2
g�1, respectively, obtained with the BETmethod. This small
decrease canbe assigned to the pore coalescence or blocking
the smaller pores during the grafting reaction [42]. The
quantity ofMB immobilized on SNwas found to be 59 mmol
g�1 similar to those obtained with silica modified with
titanium oxide [43]
3.2. Optimization of the Biosensor Components
In a previous work [39] it was demonstrated that the carbon
paste electrode modified with MB immobilized on silica gel
coated with niobium oxide (SNMB) can catalyze the
oxidation of NADH in solution with a favorable shift of
the overpotential to potentials around 0.0 V vs. SCE. The
present results show that the SNMB carbon paste electrode
can also catalyze the oxidation of NADH that is generated
from the reaction of NADþ and lactate catalyzed by LDH.
Since NADþ and mediator concentrations are constant, the
increase in the electrocatalytic current depends only on the
lactate concentration (NADH formation), and this charac-
teristic was used as the basis of the development of a
biosensor for lactate determination.
Figure 1 shows the effect of carbon paste composition on
the biosensor response for lactate, the amperometric
measurements were carried out in 0.1 mol L�1 phosphate
buffer, pH 7.0. The analytical curves obtained using carbon
paste electrodes modified with only LDH/NADþ (a) or
LDH/NADþ/SN (b) showed a negligible response at an
applied potential of 0.0 mV vs. SCE. However, for a carbon
paste electrode modified with LDH/NADþ/SNMB (c), a
visible catalytic current was observed, which provided good
sensitivity for lactate (2.1 mA cm�2 mmol�1 L). These data
confirm the efficiency of MB as an electron transfer
mediator on the electrode surface. It is observed in this
study that the response of the system is given inDj, however
the electrochemically active surface determined by G¼Q/
nFA through a cyclic voltammogram obtained with the
biosensor to 10 mV s�1 presented the result of 11.2 nmol
cm�2.
The amperometric response was also examined as a
function of the LDH loading on the carbon paste electrode.
Five carbon pastes were prepared with different amount of
LDH in the range from 0.37 up to 11.06 U of enzyme permg
of paste, while the amounts of other components were kept
constant. The sensitivity of the modified electrode was
dependent on the amount of enzyme incorporated in the
carbon paste, as shown inTable 1.An increase was observed
in the sensitivity up to 3.69 U mg�1, while for higher loads a
decrease was verified; probably the increase in the resist-
ance of the paste makes the electron transfer difficult.
Therefore, all further experiments were carried out using
3.69 U mg�1.
When an enzyme is immobilized on solid supports with
large active surface, the surface groups on the support can
induce conformational charges on the enzyme and can lead
to loss the activity. It is well known that covalent attachment
of an enzyme results in a more stable enzyme immobiliza-
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tion compared to those obtainedby physical adsorption. In
order to improve the stabilization and activity of LDH, we
studied the effect of the immobilization method and the
presence of an additive protein (BSA) on the biosensor
response. Figure 2 shows the response of the biosensor
prepared with BSA incorporated into carbon paste and
LDH without glutaraldehyde (a) and with glutaraldehyde
(b). These results show that the immobilization procedure
with glutaraldehyde/BSA gives better sensitivity and wider
linear response range, than those obtained by physical
adsorption method. The influence of BSA concentration on
the biosensor response was studied and the highest current
density was achieved incorporating 1.25% (m/m) of BSA
into the carbon paste (data not shown).
The NADþ coenzyme also plays a major role in the
biosensor mechanism. Thus, the effect on the biosensor
response using NADþ incorporated in the carbon paste or
free in solution was evaluated. This study showed that when
NADþ is incorporated into carbon paste the slopes of the
lactate calibration plots (sensitivity) are 1.5 times higher
than those obtained usingNADþ free in solution. This result
shows that the NADþ cofactor is very close to the active site
of the enzyme, sufficiently to act in an efficient form in the
enzyme reaction, leading to a high activity of the enzyme. In
addition, when NADþ was incorporated in the biosensor, a
wider linear response range was obtained.
The effect of the amount of NADþ immobilized on the
proposed biosensor response was also evaluated. A plot of
the biosensor sensitivity as a function of the NADþ
percentage in the carbon paste is presented in Figure 3. As
can be observed, the response increases sharply with
increasingNADþ percentages until 0.25% (m/m); for higher
NADþpercentages, the sensitivity decreases.Basedon these
results, 0.25% of NADþ (m/m) or 200 mg of NADþ, was
employed in the development of all the subsequent bio-
sensors. Although NADþ is highly soluble, no decrease in
the response was observed. This behavior suggests that,
during the glutaraldehyde reaction for enzyme immobiliza-
tion, the NADþ was efficiently occluded into the polymeric
net formed with BSA, LDH and glutaraldehyde, avoiding
the NADþ leaches out from the carbon paste. In addition,
previous studies [39, 44] using this kind of modified
electrode showed that NADH and mediator can form a
stable charge-transfer complex with a low tendency to
dissociation.
The decrease in the sensitivity observed in Figure 3 can be
explained due to a competition for the active site of the
enzyme by the NADþ and substrate. Thus, increasing the
NADþ amount can difficult the substrate to reach the active
site of the enzyme.
3.3. Optimization of the Operating Conditions
Biosensor response for lactate determinationusingdifferent
buffers (phosphate, Hepes, Tris and Pipes) at same pH and
concentration was performed. The investigation of the
supporting electrolyte effect pointed out to phosphate as the
Fig. 1. Effect of carbon paste composition on the biosensor
response for lactate obtained for carbon paste electrodes modified
with A) LDH/NADþ; B) LDH/NADþ/SN; and C) LDH/NADþ/
SNMB. Potential step to 0.0 mV vs. SCE, 0.1 mol L�1 phosphate
buffer solution (pH 7.0).
Fig. 2. Analytical curves obtained using the biosensor built with
BSA and LDH immobilized by physical adsorption (a) and by
cross-linking with glutaraldehyde (b). [NADþ]¼ 0.1875% (m/m),
[LDH]¼ 3.69 U mg�1 and [BSA]¼ 0.625% (m/m).
Table 1. Effect of LDH loaded on the biosensor sensitivity for
lactate determination. Potential step to 0.0 mV vs. SCE, 0.1 mol
L�1 phosphate buffer solution (pH 7.0). Sensitivity obtained by
full calibration curves. [NADþ]¼ 0.1875% (m/m) and [BSA]¼
0.625% (m/m).
LDH loading on the CPE
(U mg�1)
Sensitivity
(mA cm�2 mmol�1 L)
0.37 0.24
1.84 0.77
3.69 2.10
7.38 1.92
11.06 1.78
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best buffer system. A possible explanation for this behavior
is the complex formation between NADH and phosphate,
like an adduct, making the NADH oxidation easier [45],
while the other buffers did not affect the process.
The influence of pH on the biosensor response for lactate
was examined in the pH range between 6.0 and 8.5. As is
shown in Table 2, the current density is enhanced by
increasing the solution pH from 6.0 to 7.5, reaching a
maximum value for pH between 7.5 and 8.0. This pH range
reflected optimum conditions for both the enzymatic and
mediated electrochemical reactions in the carbon paste
electrode.
The applied potential has a strong effect on the biosensor
response. Thus, the potential dependence was examined in
order to find an optimum operational condition for lactate
determinations. As shown in Figure 4, the amperometric
response for lactate starts around �250 mV vs. SCE,
reaching higher currents when the applied potential is
increased. The response rises sharply and reaches a max-
imum current intensity at �50 mV, keeping this plateau
until 100 mV vs. SCE. In this way, 0 mV vs. SCE was chosen
as the optimum working potential since such potential
achieves a high current intensity and it is less susceptible to
the interference of easily oxidizable and reducible species.
Thus, this biosensor is less vulnerable to the interference
from several compounds commonly present in real complex
samples. In addition, at these potentials, lownoise levels and
low background currents were observed.
3.4. Analytical Curve for Lactate
TheLDHmodified carbonpaste based biosensor showed an
excellent sensitivity for lactate in a wide linear response
range. Figure 5 shows a strictly linear calibration curve
obtained from 0.1 to 14.0 mmol L�1 of lactate in 0.1 mol L�1
phosphate buffer at pH 7.8. This analytical curve was
adjusted by the equations:
Dj (mA cm�2)¼ 0.78 (�0.16)þ 2.41 (�0.02) [lactate]
(mmol�1 L), r2¼ 0.998 for n¼ 28.
Detection limits around 6.5� 10�6 mol L�1 lactate could be
estimated considering 3sB. This value is very far of the first
concentration measures and therefore, we considered 1�
10�4 mol L�1 as the quantification limit of the proposed
biosensor.
This modified electrode presented an excellent repeat-
ability, with a relative SD of 1.9% for a series of seven
successivemeasurements of a 0.5 mmol L�1 lactate solution.
Furthermore, the biosensor showed an excellent opera-
tional stability, as verified by data from repetitive measure-
ments recorded at 10 min intervals over a prolonged period,
where, around95%of the initial activitywasmaintainedafter
250 determinations (data not shown). Moreover, the pro-
posed biosensor also presented an excellent storage stability,
whichallowedmeasurements formore than2.5months,when
the biosensors were stored in a refrigerator, in a dried form.
Fig. 3. LDH/NADþ/SNMB-based biosensor sensitivity as a
function of the NADþ mass incorporated into the carbon paste.
Potential step to 0.0 mV vs. SCE, 0.1 mol L�1 phosphate buffer
solution (pH 7.0). Sensitivity obtained by full calibration curves.
[BSA]¼ 1.25% (m/m) and [LDH]¼ 3.69 U mg�1.
Table 2. 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 potential of 0 mV vs. SCE.
[NADþ]¼ 0.25% (m/m), [LDH]¼ 3.69 U mg�1 and [BSA]¼
1.25% (m/m)
pH Dj (mA cm�2)
6.0 0.19� 0.05
6.5 0.42� 0.05
7.0 1.53� 0.04
7.5 1.65� 0.04
8.0 1.67� 0.04
8.5 1.41� 0.05
Fig. 4. Dependence of the applied potential on the response of
LDH/SNMB-based biosensor. Lactate concentration¼ 5.0�
10�4 mol L�1, 0.1 mol L�1 phosphate buffer solution (pH 7.8).
[NADþ]¼ 0.25% (m/m), [LDH]¼ 3.69 U mg�1 and [BSA]¼
1.25% (m/m)
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The biosensor response time was very short and thischaracteristic is excellent considering that it is a carbon
paste electrode. Probably the design and procedure used to
construct the electrode, packed it so well that it become
difficult for the solution to diffuse through the paste and this
may contribute to its behavior [46]. In addition, the
biosensor gives a rapid response to dynamic changes in the
lactate concentration, when tested in a solution with a high
concentration (10 mmol L�1) of lactate followed by a low
one (1 mmol L�1), observed no memory effect (data not
shown). The linear response range, detection limit and,
especially, the stability presented by the proposed biosensor
are much more superior than the majority of the ampero-
metric biosensors for lactate described in the literature [47 –
54].
Reproducibility between different electrodes (n¼ 4) was
also estimated and the response of biosensor at the same
lactate concentration (1 mM) was 1.69� 0.02 mA cm�2.
3.5. Analysis of Biological Samples
The favorable characteristics presented by the proposed
biosensor allowed its application for the direct determina-
tionof lactate in real samples.Hence, theperformanceof the
biosensor was tested by applying it for the determination of
lactate in biological samples (blood plasma), before physical
exercise (sample 1) and after a physical exercise with short
duration and high intensity (sample 2).
Table 3 summarizes the concentrations found by the
biosensor and reference method (commercial Kit). As can
be observed, the data showed an excellent correlation
between the results obtained with the biosensor and with
reference method (spectrophotometric), with accuracy
higher than 95%. This performance presented by the
developed biosensor indicates that it should be highly
selective to the lactate.
Figure 6 shows the stability study of the proposed
biosensor in operational conditions (measured in biological
sample). As can be observed, around 95% of the initial
response was maintained after 20 determinations.
On the other hand, the proposed amperometric biosensor
presents some advantages in relation to the standard
method, such as analysis time and the chemicals required
to prepare biosensor is lower than those used for standard
method. Moreover, the biosensor preparation in one step
associated to the possibility of the coenzyme and enzyme
regeneration makes the cost per analysis cheaper.
4. Conclusions
TheMB adsorbed on silica gel modified with niobium oxide
provided an excellent electro catalyst property for NADH
oxidation. This material was very useful for a simple and
effective way to develop biosensors for lactate determina-
tion. The proposed LDH-based biosensor exhibited an
excellent operational and storage stability. The experiments
described above illustrate the ability to employ this bio-
sensor for lactate detection in real samples, without adding
any reagent, presenting good sensitivity, selectivity, speed,
Fig. 5. Typical analytical curve obtained with the LDH/NADþ/
SNMB-based biosensor. Potential step to 0.0 mV vs. SCE, 0.1 mol
L�1 phosphate buffer solution (pH 7.8). [NADþ]¼ 0.25% (m/m),
[LDH]¼ 3.69 U mg�1 and [BSA]¼ 1.25% (m/m). Inset the
current – time response curve.
Table 3. Determination of lactate in plasma samples.
Sample Reference method
(mmol L�1)
Biosensor
(mmol L�1)
Relative
difference (%)
#1 1.68� 0.03 1.77� 0.04 5
#2 12.42� 0.23 12.66� 0.31 2
Fig. 6. Operational stability obtained for the biosensor in plasma
sample; potential step to 0.0 mV vs. SCE, 0.1 mol L�1 phosphate
buffer solution (pH 7.8). [NADþ]¼ 0.25% (m/m), [LDH]¼ 3.69 U
mg�1 and [BSA]¼ 1.25% (m/m).
1213Amperometric Biosensor for Lactate
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precision, which are intended for any analytical method-
ologies.
5. Acknowledgement
The authors thank FAPES for the financial support.
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