ABSTRACT
A
biosensor was developed for spectrophotometric determination of
glucose concentrations in real samples of orange juice energetic
drinks, and sport drinks. The biosensor consisted of glucose oxidase
(GOD) and horseradish peroxidase (HRP) immobilized onto polyaniline
activated with glutaraldehyde (PANIG). Immobilization parameters were
optimized for GOD, and maximum immobilization yield was 16% when 5.0 mg
of PANIG and 8.9 U prepared in 0.1 mol.L-1 sodium
phosphate buffer (pH 7.0) reacted for 60 minutes at 4 °C with gentle
stirring. The linear operational range for glucose determination
using optimized operational parameters was between 0.05 and 6.0 mg.mL-1
with a very good reproducibility of response. The results obtained
in the biosensor were compared with those obtained using free enzymes
(commercial kits) and then validated through statistical analysis
using the Tukey test (95% confidence interval).
RESUMO
Foi
desenvolvido um biossensor espectrofotométrico para a determinação
da concentração de glicose em amostras reais de suco de laranja,
energéticos e bebidas esportivas. O biossensor consistiu de glicose
oxidase (GOX) e peroxidase (HRP) imobilizadas em polianilina ativada
com glutaraldeído (PANIG). Os parâmetros de imobilização foram
otimizados para a GOX e o rendimento máximo de imobilização obtido
foi de 16% para 5 mg de PANIG e 8,9 Unidades de Enzima (UE) preparadas
em tampão fosfato 0,1 mol.L-1 (pH 7,0) com tempo de reação
de 60 minutos a 4 °C, com agitação leve. Utilizando os parâmetros
otimizados, encontrou-se uma faixa linear para a determinação de
glicose entre 0,05 e 6,0 mg.mL-1, com boa reprodutibilidade
de resposta. Os resultados obtidos foram comparados com resultados de
enzimas livres (kits comerciais), sendo validados através de análise
estatística pelo teste de Tukey (95% de significância).
Starch
is one of the biomolecules that appears frequently in food
composition due to its physical properties, and because it acts as a
source of glucose and therefore of energy (DEMIATE; KONKEL; PEDROSO,
2001; WEBER; COLLARES-QUEIROZ; CHANG, 2009). The proliferation of
microorganisms in foodstuffs is directly dependent on their ability
to hydrolyze starch releasing glucose that will be the primary source
of energy for growth. In this case, increase in glucose concentration
may be an important and early indicator of contamination.
The
determination of glucose concentration is also particularly
important in some food products since glucose causes browning during
dehydration and long-term storage, mainly due to the Maillard
reaction. This is a major obstacle in the manufacture of dehydrated
egg powder (D'SOUZA; GODBOLE, 1989), and it is also a serious problem
in hydrolysis of high molecular weight dextrans to oligosaccharides,
utilized in the production of pharmaceutical products. The resulting
colored compounds are not compatible with pharmaceutical and food
rules for formulation acceptability (MISLOVICOVÁ; MICHÁLKOVÁ;
VIKARTOVSKÁ, 2007). Therefore, the quantitative determination of
glucose constitutes an important measurement in food product quality
control.
Numerous
methods have been reported for glucose analysis in food. However,
most of the current adopted methods are time consuming or costly
(AQUINO et al., 2004; WU et al., 2004; DRUZIAN; DOKI; SCAMPARINI,
2005; YILDIZ et al., 2005). Currently, several devices are available
for precise and prompt glucose measurements in the clinical area, but
these devices are frequently designed to operate under blood serum
conditions. Development of fast, cheap, practical, and selective
methods for detecting glucose in food is still a research area that
brings together efforts from chemistry to clinical analysis.
Enzymatic
determinations of glucose involve glucose oxidase (E.C. 1.1.3.4.)
(GOD) and horseradish peroxidase (E.C. 1.11.1.7) in a sequence of
redox reactions. Glucose oxidase as a free enzyme has been used in
the measurement of glucose in foods and beverages (ÇIL et al., 2007;
WILSON; TURNER, 1992; SARTINI et al., 1998) even though it represents
an important cost component of a product (WU et al., 2004). To overcome
this inconvenience, GOD has been immobilized on various supports
with the purpose of its economic reuse (MISLOVICOVÁ; MICHÁLKOVÁ;
VIKARTOVSKÁ, 2007; ENDO et al., 2006; FERREIRA; FIORITO; OLIVEIRA
JUNIOR, 2004; GÜRSEL et al., 2003). Examples of the use of
immobilized GOD in food production are the preparation of gluconic
acid (RAMACHANDRAN et al., 2006; BAO et al., 2004; FIEDUREK, 2001),
removal of oxygen from beer (HARTMEIER; WILLOX, 1981), and the
removal of glucose from eggs (SISAK et al., 2006; SANKARAN; GODBOLE;
D'SOUZA, 1989; D'SOUZA; GODBOLE, 1989). When immobilized in
biosensors, GOD is also used for the removal of glucose from pentosan of
wheat flour (HOUBEN; RUIJTER; BRUNT, 1997), and health control of
farm fish (ENDO et al., 2006). In spite of its wide application, the
most common and serious problem of GOD biosensors is their lack of
stability because of the intrinsic nature of the enzyme (KANG et al.,
2007).
In
a previous study, we showed a spectrophotometric device for hydrogen
peroxide measurement using horseradish peroxidase (HRP) immobilized
on polyaniline (FERNANDES et al., 2005). This device was used to
measure glucose in a combination with free GOD. In the present paper,
the development of a cheap and stable biosensor containing HRP and
immobilized GOD and its use for spectrophotometric determination of
glucose in real samples of sport drinks, soft drinks, and orange juice,
is described.
2 Materials and methods
2.1 Reagents
Horseradish
peroxidase (HRP) type VI was purchased from Sigma Chemical Co. (St.
Louis, MO, USA). Glucose oxidase (GOD) 90 U.mg-1, hydrogen
peroxide, and aniline were obtained from E. Merck (Darmstadt,
Germany). Aniline was distilled under vacuum obtaining a colorless
liquid. Energil C and orange juice were produced by Tampico - Tampico
Beverages Inc., Chicago - IL, EUA. On Line and Fly horse were produced
by AmBev - GlobalBev, Marathon by Fratelli Vita - AmBev - GlobalBev.
Red bull and Gatorade were produced by Quaker Oats Company. All other
reagents were analytical grade and were used without further
purification. Solutions were prepared with deionized water.
2.2 Polymer synthesis and activation
Polyaniline
(PANI) was chemically synthesized as described previously
(FERNANDES et al., 2003) using ammonium persulphate as the oxidizing
agent. The black/green powder obtained was activated with a 2.5%
(v/v) glutaraldehyde solution, under reflux, for 2 hours to obtain
glutaraldehyde-modified polyaniline (PANIG). After exhaustively
washing with 0.1 mol.L-1 phosphate buffer, pH 6.0, the
powder was dried under dynamic vacuum and stored at room temperature
until its use for enzyme immobilization.
2.3 Immobilization and assay
The immobilization of HRP was carried out as described previously (FERNANDES et al., 2003) resulting in 8.2 U HRP.mg-1 PANIG, which represents a 24.3% yield.
The
optimum immobilization conditions of glucose oxidase (PANIG-GOD)
were obtained varying the enzyme and polyaniline (PANIG)
concentration, the pH, and the time of the immobilization reaction.
The reactions were performed at 4 °C. After immobilization, PANIG-GOD
was washed with 0.1 mol.L-1 phosphate buffer pH 7.0 to remove unbounded enzyme molecules.
Measurements
of GOD immobilization were performed by adding 2.8 mL of freshly
prepared substrate solution containing 41.05 mmol.L-1 phenol, 0.6 mmol.L-1 4-aminoantipyrine, both prepared in 0.1 mol.L-1
phosphate buffer, and pH 7.0 to PANIG-GOD. The reaction was started
by the addition of 20 µL of glucose solution (0.05 to 6 mg.mL-1)
which were left to occur for 10 minutes. The product formed was
analyzed using a spectrophotometer at 510 nm. All measurements were
performed in triplicates, and the results were presented as mean and
standard deviation.
2.4 Reactor design
The
biosensor was constructed as an air-lift reactor consisting of
5.0 mg of PANIG-HRP and 5.0 mg of PANIG-GOD assembled into a
polypropylene reaction camera connected to an air pump, an upper
connection for substrate input and a lower valve for product exit.
The products were pumped through a detection system consisting of a
spectrophotometer connected to a recorder. Optical density was recorded
every 5 seconds at 510 nm in a glass cell with 1.0 mm optical path
length (Figure 1).
The relationship between PANIG-HRP and PANIG-GOD amounts in the
reaction camera were optimized to obtain the best performance of the
system.
2.5 Operational range for glucose determination and operational stability of the biosensor
The
system was operated at room temperature. The operations consisted of
filling the reaction camera containing the immobilized enzymes with
substrate solution (2.2). 20 µL injections of increasing
concentrations of glucose samples (0.05 a 6.0 mg.mL-1)
started the reactions, which were left to proceed for 10 minutes.
Next, the exit valve was opened, and the product formed was pumped to
the detection system. The residence time was tested from 1 to
30 minutes. Only measurements higher than 0.4 EU was considered to
establish the inferior limit of detection. Readings below this value
were considered as noise.
The
operational stability of the biosensor was tested by sequential
injections of glucose samples followed by washes with phosphate
buffer.
2.6 Determination of glucose in real samples
The
amount of glucose in real samples was determined using biosensor.
The tests were performed in triplicates, and the results were
presented as mean and standard deviation. The operations consisted of
filling the reaction camera containing the immobilized enzymes with
the substrate solution (2.2). Injections of 20 µL of the beverages
samples started the reactions which were left to proceed for 10 minutes.
Next, the exit valve was opened, and the product formed was pumped
to the detection system. The data obtained using the biosensor were
compared with the glucose amounts determined via glucose oxidase
method using free GOD and HRP (HENRY, 1996). Total sugar
concentration was measured using the Dubois (phenol sulphuric) method
(1956).
2.7 Statistical analysis
All
experiments were performed at least three times in triplicate with
three independent samples. The analysis of variance (ANOVA) and
comparison between means values were applied to establish differences
using the Tukey test (confidence interval 95%). All analyses were
carried out using the Statistica 6.0 software.
3 Results and discussion
3.1 GOD immobilisation
The
immobilization parameters were optimized for GOD resulting in a
maximum immobilization yield of 16% when 5.0 mg of PANIG and 8.9 EU
prepared in 0.1 mol.L-1 phosphate buffer, pH 7.0, were left to react for 60 minutes, at 4 °C under gently stirring. Figure 2
shows that pH seems to be an important immobilization factor because
variations of one pH unit resulted in a significant increase in the
immobilized enzyme content. This variation was probably caused by the
state of ionization of the lateral chains of the lysine residues
present on the exposed surface of GOD since these are the targets for
reaction with the carbonyl groups of glutaraldehyde to form Schiff
bases (ARTIGO LU). Furthermore, as can be seen in Figure 3,
increases in enzyme concentration resulted in increases in the
amount of immobilized enzyme. Efficiency of immobilization in the
optimized conditions was higher than those found by Du et al. (2009),
who reported a yield of 3.36% when GOD was immobilized in Poly
(St-GMA-NaSS) microspheres with and without BSA-spacer.
3.2 Operational range for glucose determination
Figure 4a, b, c
shows the response obtained in the biosensor for three independent
glucose injections. The operational range for glucose determination
was stated from 0.05 to 6.0 mg.mL-1. As can be seen, the
system is able to determine glucose with high reproducibility and
linearity of response (a, r = 0.997; b, r = 0.995, and c, r = 0.996).
The results obtained showed that the operational range for glucose determination (0.05 to 6 mg.mL-1)
used in this study was larger than that reported by Duong and Rhee
(2007), who obtained values of the detection limit between 0.10 -
0.52 mg.mL-1.
3.3 Long-term stabilities of GOD/HRP biosensor
To evaluate long-term stabilities of the biosensor, the responses were monitored during continuous use. As can be seen in Figure 5,
the biosensor was used in 25 cycles for glucose determination
without any loss in sensitivity. This finding is very important since
it combines the stability of two immobilized enzymes: HRP and GOD. A
preliminary study showed that HRP immobilized onto polyaniline was
very stable for hydrogen peroxide determination (FERNANDES et al.,
2005). Several authors reported stability of enzyme biosensors
containing immobilized GOD (ÇIL et al., 2007; MISLOVICOVÁ;
MICHÁLKOVÁ; VIKARTOVSKÁ, 2007; YILDIZ et al., 2005; FERREIRA;
FIORITO; OLIVEIRA JUNIOR, 2004; GÜRSEL et al., 2003). The variation
observed in this parameter is very high, and as such it is possible
to find stabilities from 45% of initial activity after 5 days of use
and storage to 90% of initial activity after 45 cycles of use. Based
on these data, the biosensor described here presents good stability,
especially considering the ease of the polymer synthesis,
immobilization, and the operation of the device.
3.4 Determination of glucose amount in real samples
Several
sport and energetic drinks as well as orange juice were analyzed for
their glucose contents using a GOD/HRP biosensor and also by a free
enzyme system. Table 1 shows the results of glucose determination in the biosensor and the use of the free enzymes.
As
can be seen, there was a close relationship between the values found
for the glucose measurements in the biosensor and those obtained
with free enzymes. Slightly higher variations were observed among the
total amounts of carbohydrates informed by the manufacturer and
those obtained by the phenol-sulphuric method.
One
interesting finding of this study was the absence of interference in
the measurements. The composition of energetic beverages and soft
drinks presents several compounds which can interfere in the
performance of enzymes. Compounds such as citric acid, ascorbic acid,
and several others may interfere in the reactions catalyzed by both
GOD and HRP. Other components of these drinks may be oxidized by
hydrogen peroxide produced in the HRP reaction hindering the GOD
reaction. Particularly, biosensors can suffer from polymer
interference if the support used for immobilization act as an
adsorbing component. The absence of interference in the case of
PANIG-GOD-HRP is a very interesting finding.
4 Conclusion
This
study showed that GOD and HRP can be successfully immobilized in
PANIG polymer, and that the biosensor resulting from this procedure
represented a cheap, fast, and practical alternative to glucose
determination in the samples tested. The biosensor showed high
reproducibility of response and high stability during repeated use.
Another important characteristic of this biosensor was the wide range
of detection. The absence of interference of the components in the
samples, especially ascorbic acid in the orange juice and citric acid
in all the other beverages, was a very important finding of this
research opening up the possibility of a wide range of application
possibilities of this biosensor.
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Fonte: http://www.scielo.br
