Amino acid intercalated montmorillonite: Electrochemical biosensing applications

Article abstract of DOI:10.1039/c4ra07026g

The present work is the first that includes the use of glycine (Gly), lysine (Lys) and glutamic acid (Glu) modified clay mineral matrices in the biosensors. For this purpose, initially, Gly, Lys and Glu were intercalated with montmorillonite (Mt), thus, various modified Mts were obtained. These modified materials were then characterized via X-ray diffraction, Fourier transform infrared spectroscopy, zeta potential and thermal gravimetric analysis (TGA/DTG) and scanning electron microscopy (SEM). In order to investigate the applicability of amino acid modified clay minerals in biosensor areas, glucose oxidase (GOx) was selected as the model enzyme and a GOx based biosensor was prepared. After immobilizing the enzyme with amino acid modified Mt onto a glassy carbon electrode, working conditions like pH and modifier type were optimized. Among the modified Mts, Gly-Mt was the optimum clay mineral type and pH 4.0 was the optimum pH value. Then analytical characteristics were examined under optimum experimental conditions. The linear range of optimum sensor design was 0.1-1.0 mM within the kinetic parameters of an immobilized enzyme K_m^app = 0.7 mM, I_max = 107.8 nA. Finally, the developed biosensor was applied to real samples where the results were compared with a spectrophotometric reference method.

Full text of DOI:10.1039/c4ra07026g

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Amino acid intercalated montmorillonite:
electrochemical biosensing applications
Cite this: RSC Adv., 2014, 4, 50107
a
a
b
c
a
Filiz Demir, Bilal Demir, Esra E. Yalcinkaya, Serdar Cevik, Dilek Odaci Demirkol,
c
a
Ulku Anik and Suna Timur*
The present work is the first that includes the use of glycine (Gly), lysine (Lys) and glutamic acid (Glu)
modified clay mineral matrices in the biosensors. For this purpose, initially, Gly, Lys and Glu were
intercalated with montmorillonite (Mt), thus, various modified Mts were obtained. These modified
materials were then characterized via X-ray diffraction, Fourier transform infrared spectroscopy, zeta
potential and thermal gravimetric analysis (TGA/DTG) and scanning electron microscopy (SEM). In order
to investigate the applicability of amino acid modified clay minerals in biosensor areas, glucose oxidase
(GOx) was selected as the model enzyme and a GOx based biosensor was prepared. After immobilizing
the enzyme with amino acid modified Mt onto a glassy carbon electrode, working conditions like pH and
modifier type were optimized. Among the modified Mts, Gly-Mt was the optimum clay mineral type and
pH 4.0 was the optimum pH value. Then analytical characteristics were examined under optimum
Received 12th July 2014
Accepted 25th September 2014
experimental conditions. The linear range of optimum sensor design was 0.1–1.0 mM within the kinetic
app
parameters of an immobilized enzyme K
m
¼ 0.7 mM, Imax ¼ 107.8 nA. Finally, the developed biosensor
DOI: 10.1039/c4ra07026g
was applied to real samples where the results were compared with a spectrophotometric reference
method.
www.rsc.org/advances
1
0,11
utilized for encapsulant material in intercalation processes.
1
Introduction
3–12
13
In this way, alkylammonium salts,
polymers and amino
14
In the last decade, there has been a growing interest towards acids (mostly hydrophobic amino acids) have been used as the
combining inorganic clay nanocomposites within enzymes or intercalating agents. On the other hand, these modiers have
cells by taking advantage of the physical and chemical proper- enabled thermostability and improved mechanical strength to
ties. These materials can be used in many biological applica- clay minerals. Therefore, organo-modication of clay minerals
1
–7
tions including biosensors, drug release and cell adhesion.
has been drawing interest for clay modied electrode prepara-
The crystal structure, morphology and chemical composition of tion and enzyme immobilization. However, clay mineral
clays offer ion exchange capacity, good thermal, mechanical modied enzyme electrodes can suffer from enzyme leaching
and chemical stabilities and advanced adsorption properties. during the application. Hence, by adding the glutaraldehyde
Because of this, they can be used as a suitable immobilization (GA) and bovine serum albumin (BSA) to the biolm, a robust
8
15,16
platform for a facile biosensing transducer.
Phyllosilicates are 2 : 1 layered structure consisting of an
octahedral sheet which is predominantly occupied by trivalent onto clay minerals, adsorption capacities of amino acids and
biosensing platform can be obtained.
In many studies based on the intercalation of amino acids
3
+
2+
or divalent cations (i.e. Al or Mg ) sandwiched between two their detailed characterization related to pH, temperature,
tetrahedral siliceous sheets. Vermiculites and smectites that interlayer spaces have been conducted with Fourier transform
include montmorillonite (Mt), nontronite and laponite are infrared spectroscopy, X-ray diffraction, thermogravimetric
17–22
typical clay minerals and belonging to phyllosilicate family. As analysis and zeta potential.
However, it can be seen that not
cationic clay mineral, Mt exhibits some unique characteristics so much work reported in the literature for the application of
9
like intercalation, swelling and strong adsorption. In addition amino acid modied clay minerals, particularly in biosensor
to that, Mt can be an appropriate natural silicate which can be areas. Avinash et al. (2011) reported the unnatural amino acid
(
5-aminovaleric acid) modied Mt and their use in tissue
23
a
engineering via forms biocomposites with hydroxyapatite.
Ege University, Faculty of Sciences, Biochemistry Department, 35100-Bornova, Izmir,
Also in another study, Wang et al. (2013) synthesized and
Ege University, Faculty of Sciences, Chemistry Department, 35100-Bornova, Izmir, characterized the Ga -salicylidene-amino acid (glycine,
Turkey. E-mail: suna.timur@ege.edu.tr
b
3
+
Turkey
alanine, tyrosine, phenylalanine, aspartic acid and glutamic
acid, respectively) schiff base complexes with layered double
c
Mugla Sitki Kocman University, Faculty of Sciences, Chemistry Department, Kotekli,
Mugla, Turkey
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hydroxides to evaluate potential applications of drug delivery dispersions was the average of the data obtained from at least
24
system for antimicrobial activity.
10 experiments. The surface morphology was monitored with a
Present study consists of the intercalation of Mt with glycine, FEI Quanta250 FEG scanning electron microscope (SEM). An
lysine and glutamic acid amino acids, their detailed character- accelerating voltage of 5.0 kV was applied for each sample, and a
ization and application to biosensors. In biosensing experi- spot size of 3 was used. All of the samples were dried in a
ꢁ
ments, amino acid modied Mts have been used as vacuum oven before the analyses at 50 C for 2 days.
immobilization materials for glucose oxidase (GOx) adsorption
onto Mt layers via GA + BSA support as proposed in previous 2.2 Modication of organo-montmorillonite
work based on histidine modied Mt based Laccase
In order to have a successful development of clay mineral-based
25
biosensor. Aer the electrochemical characterization, opti-
mization and determining the analytical parameters, the most
efficient sensor design was applied to real samples.
biosensors, it is necessary to chemically modify a naturally
hydrophilic silicate surface to an organophilic one so that it can
be compatible with a chosen enzyme. The modied Mt was
prepared by the method of ion exchange. Sodium-Mt powder
(0.5 g) was dispersed in 200 mL deionized water under stirring
with a Heidolph magnetic stirrer for about 24 h. The glutamic
acid (Glu), lysine (Lys) and glycine (Gly) amino acids were
separately dissolved in 20 mL deionized water. The amounts of
amino acids were equivalent to 2 times the CEC of Mt. These
intercalating agents were rstly protonated with 1.0 M HCl
aqueous solutions to adjust the pH to 2.0–3.0. Then, it was
slowly dropped into the Mt dispersion for stirring 24 h at room
temperature. The modied Mts (Glu-Mt, Lys-Mt and Gly-Mt)
were separated by ltration and washed several times with
2
Material and method
2.1 Reagents and apparatus
ꢀ1
Glucose oxidase (GOx; EC 1.1.3.4, 50 U mg
protein),
D-(+)-glucose, bovine serum albumin (BSA), glycerol, glutaral-
dehyde (GA) solution (25%, v/v) were purchased from Sigma
Chem. Co. (St. Louis, MO, USA; http://www.sigmaaldrich.com).
The clay mineral used for the experiments was obtained from
West Anatolia. It was puried from bentonite before use.
Particles, 2 mm in diameter were obtained by sedimentation
ꢀ
1
(
ion-exchange capacity; 92 meq. 100 g ). Amino acids (glycine,
deionized water until no chloride ions were detected by AgNO
3
glutamic acid and lysine) were purchased from Sigma Chem.
Co. (St. Louis, MO, USA). All other chemicals were used of
reagent grade.
solution. The obtained organic Mt was dried in a vacuum oven
ꢁ
at 50 C for 2 days. Aer the 2 days, the weight loss has not been
observed.
PalmSens Potentiostat (Palm Instruments, Houten, Nether-
lands) and FRA 2 m-AUTOLAB Type III (ECO CHEMIE Instru-
ments B.V., Netherlands) were used for amperometric,
impedimetric and voltammetric measurements. A three elec-
trode cell where clay mineral modied electrodes as working
electrode, Ag/AgCl electrode with 3.0 M KCl as a reference
electrode and platinum (PT) as the counter electrode was used.
Pharmacia LKB Novaspec II spectrophotometer was used for the
spectrophotometric measurements.
2
.3 Fabrication of A.A-Mt/GOx biosensors
Initially, GCE was polished with alumina slurry (0.05 mm sized)
and then bath sonication was applied for 5 min in etha-
nol : distilled water (1 : 1) solution. To construct the enzyme
ꢀ
1
electrode, 5.0 mL of Mt solution (1.0 mg mL ), 0.5 mg GOx
ꢀ
1
(which is equal to 25 U), 2.5 mL of BSA (1.0 mg mL ) and 2.5 mL
of GA (5.0%) were mixed. Aerwards, the mixture was
spread over the electrode and allowed to dry at room tempera-
ture for 1 h.
For the characterization, all samples were rst examined
with X-ray diffraction (XRD) Spectrometers (Philips E'xpert Pro;
All reagents were prepared in phosphate buffer (50 mM, pH
ꢀ
Cu-Ka radiation, l ¼ 1.541 A) to determine the interlayer spaces
7.0). Prior to measurements, each daily prepared enzyme elec-
of Mt and Mod-Mts. The XRD spectra were obtained at a scan-
ꢀ1
trode was washed with distilled water to eliminate unbounded
enzyme.
ning rate of 2.00 min and the 2q angles ranged from 5.00–
0.00. The basal spacing values of pure Mt and Mod-Mts were
8
calculated by Bragg's law. Fourier transform infrared (FTIR)
spectra of clay minerals were recorded using a Perkin Elmer
2.4 Electrochemical measurements
Pyris 1 FTIR Spectrometer on KBr plates. Thermal degradation All amperometric experiments were performed in working
was studied with a thermogravimetric analyzer (Perkin Elmer buffer solution (10 mL). Electrodes were rinsed with distilled
Pyris 1 TGA/DTA) by heating the samples from ambient water and kept in sodium acetate buffer (50 mM, pH 4.0) for
ꢁ
ꢁ
ꢀ1
temperature to 1000 C at 10 C min under a 10 bar dry air GOx biosensor at least 3 min aer each trial. Enzyme immobi-
atmosphere. The electrokinetic properties of clay minerals were lized electrodes were initially equilibrated in the buffer and
determined by measuring the zeta-potential of particles with a then, the substrate (glucose) was added to the reaction cell.
Zeta-Meter 3.0+ (with a Zeiss DR microscope, GT-2 type quartz
During the measurement of GOx electrode, an enzymatic
cell, molybdenum-cylinder anode, and platinum-rod cathode reaction proceeds on two half reactions namely a reduction and
electrode). The clay mineral samples were stirred overnight in a following oxidative step. In the rst step, GOx which is a
the deionized water to obtain well dispersed particles. The zeta dimeric protein containing one tightly bound avin adenine
potential of the modied Mt dispersions was estimated from dinucleotide (FAD) per monomer as cofactor, catalyzes the
the measured electrophoretic mobilities by employing the oxidation of b-D-glucose to D-glucono-d-lactone, which is spon-
26
Smoluchowski equation. The zeta potential assigned to the taneously converted to gluconic acid and in the second step, the
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2 2 2
reduced GOx(FADH ) is reoxidized by oxygen to yield H O . In
GOx, FAD acts as an electron acceptor, and it is reduced to
FADH ; the FADH is then oxidised by the nal electron
2
2
acceptor, molecular oxygen, with the oxygen being reduced to
27
2 2
hydrogen peroxide (H O ). The decrease in the amount of
oxygen in bioactive layer is monitored at ꢀ0.7 V with respect to
Ag/AgCl electrode as a result of enzymatic activity. The
biosensor responses were registered as the current signal (nano
or m-A). The buffer was refreshed aer each measurement. All
chronoamperometric measurements were performed 3 times.
Data were presented as the mean ꢂ standard deviation (S.D).
Cyclic voltammetry (CV) experiments and electrochemical
impedance spectroscopy (EIS) measurements were performed
on a FRA 2 m-AUTOLAB Type III electrochemical measurement
system from ECO CHEMIE Instruments B.V., (Netherlands)
driven by NOVA soware (http://www.ecochemie.nl). EIS
measurements were performed in sodium phosphate buffer
Fig. 1 FTIR spectra of (a) Mt, (b) Glu-Mt, (c) Lys-Mt and (d) Gly-Mt.
3ꢀ/4ꢀ
(50 mM, pH 6.5) containing 5.0 mM [Fe(CN)6]
with a
frequency range from 0.1 Hz to 100 kHz at 0.2 V. CV measure-
ments were conducted in the potential range of ꢀ0.4 V to 0.8 V
with a 0.0244 V step potential and using only clay mineral lm
2
9
results. In addition to these bands, the bands around 1400
ꢀ1
cm are attributed to the presence of N–H on the structure.
(without biological compound or cross-linker) in the presence
The interlayer space and the crystalline conguration of the
clay minerals can be identied by XRD. The basal spacing
values of modied Mts were compared to pristine Mt to indicate
the presence of amino acid molecules. Fig. 2 shows the XRD
patterns of Mt and amino acid modied Mts (Glu-Mt, Lys-Mt
and Gly-Mt). According to the XRD pattern of Mt, the typical
of 0.1 M KCl used as the supporting electrolyte.
2.5 Sample application
The biosensors were used to analyse the glucose content of
some beverages. As a reference method, Trinder reagent which
is a commercial enzyme assay kit was utilized for the analysis of
the same samples spectrophotometrically. The method is based
on the oxidation of glucose to D-gluconate by GOx with the
formation of hydrogen peroxide. In the presence of peroxidase,
a mixture of phenol and 4-aminoantipyrine is oxidized by
hydrogen peroxide to form a red quinine imine dye proportional
+
XRD reection of Na -Mt related to the basal spacing between
ꢁ
the clay mineral layers appeared at 2q ¼ 7.74 . The basal
ꢀ
spacing value of Mt was calculated as d001 ¼ 11.4 A from this
ꢁ
ꢁ
ꢁ
value. For modied Mts, the peak at 7.74 shis to 6.82 , 6.76
ꢁ
and 6.02 in the spectrum of Gly-Mt, Lys-Mt and Glu-Mt,
respectively. This reection appeared at the lower angles of
the diffractogram indicated the intercalation of amino acid
molecules in the interlayer spaces of Mod-Mts. The basal
28
to the glucose concentration in the sample.
spacing of the modied Mts was calculated as d001 ¼ 12.96,
3
Results and discussion
13.06 and 14.67 ꢀA for Gly-Mt, Lys-Mt and Glu-Mt, respectively.
ꢀ
The swelling of modied Mts are 1.56, 1.66 and 3.27 A for Gly-
3.1 Characterization of organo-modied clay minerals
The intercalation of organic-modied clay minerals was char-
acterized by FTIR, XRD, and zeta potential measurements. Also,
the thermal stability of pure and modied clay minerals was
determined with TGA and DTG analysis. The nature of the
chemical groups intercalated was investigated by FTIR and their
spectra are shown in Fig. 1. In the FTIR spectrum of Mt (Fig. 1a),
ꢀ
1
the band at 3630 cm is ascribed the –OH bond corresponding
to the characteristic stretching for Al–Al–OH bond in the octa-
ꢀ
1
hedral layer of Mt. The band at around 1630 cm is related with
ꢀ1
H–OH stretching vibration. An intense peak at 1054 cm is the
characteristic absorption band for Mts corresponding to Si–O
stretching. In the FTIR spectra of Mts (Fig. 1b–d), characteristic
absorption bands for both Mt and modiers were observed. In
addition to Mt band, the carbonyl group (C]O) band of the
amino acids that proves the modication were observed at 1700
ꢀ
1
cm at all FTIR spectra and in another work that was studied
with natural amino acid intercalation of cloisite, the peaks for
–
COOH group in modied clay minerals support this FTIR Fig. 2 XRD patterns of Mt and modified-Mts.
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Mt, Lys-Mt and Glu-Mt, respectively. The increasing of basal that Gly was adsorbed mostly on the clay mineral surface and
spacing values of Mt could be seen clearly due to the increasing also intercalated to the layers of Mt easily due the its un-
of the interlayer distance of the Mt with amino acids. It is branched and compact structure. However, the percentages of
attributed that the cation exchange process between the inter- ion exchange were calculated as 13.5 and 13.4% for Glu-Mt and
layer cations of Mt and amino acid salts was effectively carried Lys-Mt, respectively by using CEC. It is evident that Glu and Lys
out and amino acids intercalated into the Mt layers.
amino acids could not intercalated to the layers of Mt easily
The thermal stability of Mt and amino acid modied Mts according to the Gly amino acid due the its branched struc-
Glu-Mt, Lys-Mt and Gly-Mt) was compared with TGA analysis. tured. Also, the thermal stability of the modied Mts was found
(
The TGA thermograms are shown in Fig. 3a. Mt exhibited about lower than that of pristine clay mineral as our previous
ꢁ
4–12
8
.4% weight loss at 1000 C because of the presence of volatile studies.
ꢁ
materials. Degradation started at 100 C due to the unbounded
The DTG thermograms of the samples have already reported
H O and continued up to about 800 C due to the chemically that the derivative of weight loss as a function of temperature in
ꢁ
2
bound H
2
O. The weight loss of the amino acid modied Mts Fig. 3b. The max decomposition peak temperature of pure Mt
ꢁ
was founded at 15.4, 17.4 and 21.5 weight% for Glu-Mt, Lys-Mt was 635 C from the thermogram of Mt. According to DTG
and Gly-Mt, respectively. The increasing of the weight loss was thermograms of Glu-Mt, Lys-Mt and Gly-Mt, the two decompo-
due to the adsorption of modiers both at the surface and sition stages of modied Mts were between 100–800 C. The rst
ꢁ
within the clay mineral layers. These results indicated that 6.0, peak corresponding to the degradation of the intercalated
ꢀ
1
9
.0 and 13.1 weight% (0.4, 0.6 and 1.7 mmol g ) of Glu, Lys and species as Glu, Lys and Gly salts into Mt interlayer spaces. The
ꢁ
Gly as modier were holding onto the Mt structure. For Gly-Mt, max degradation peak temperatures of were 310, 250 and 235 C
the percentage of ion exchange was calculated as 6.9% by using for Glu-Mt, Lys-Mt and Gly-Mt, respectively. Hence, the Glu-Mt
CEC of Mt that was lower from the TG data. This was indicated has the max degradation peak temperatures due to the its
structure. The second decomposition stages were occurred at
ꢁ
about 640 C due to the decomposition of clay mineral particles
like with the pristine Mt.
When a solid surface is in contact with an aqueous solution,
the formation of an interfacial charge causes a re-arrangement
of the local free ions in the solution. The arrangement of the
charges at the solid–liquid interface and the balancing coun-
terions in the liquid is usually referred as the electrical double
layer. The zeta potential is an indicator of the surface charge
properties of a colloid or a particle in solution and varies
depending on the surface potential and the thickness of the
electric double layer. It is usually characterized by the
measurement of the electrophoretic mobility of the colloidal
particles in dispersion which is a key technique for the deter-
30
mination of the surface properties of the particles. The zeta
potential can be used for studying and predicting colloidal
stability and particle surface charge, effectively. It is also an
important parameter for a number of applications including
characterization of biomedical polymers, electrokinetic trans-
port of particles or blood cells, sensors and biosensors,
31–33
membrane efficiency and microuidics.
zeta potential of the Mts was calculated as ꢀ42.0 ꢂ 2.24, ꢀ34.3 ꢂ
.85, ꢀ33.3 ꢂ 0.65 and ꢀ30.3 ꢂ 0.43 mV for Mt, Glu-Mt, Lys-Mt
In this study, the
0
and Gly-Mt, respectively. Aer each modication step, the zeta
potentials shied to less-negative values as a result of adsorption
of positively charged amino acid salts at the surface or interlayer
of the mineral. Gly-Mt exhibited the lowest negative zeta
potential value compared to that of the other amino acid
modied clay mineral that it is compatible with TGA results.
The surface morphologies of the Mt and amino acid modi-

ed Mts were compared by SEM analyses. The SEM micro-
graphs are shown at Fig. 4 for Mt, Gly-Mt, Lys-Mt and Glu-Mt,
respectively. It was clearly observed that the modied Mts
surfaces exhibited a rough surface aer this modication step.
The clay platelets were stacked together in a disordered pattern
to form agglomerates in some parts, and small and well-
Fig. 3 (a) TG thermograms of Mt and modified-Mts. (b) DTG ther-
mograms of Mt and modified-Mts.
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electron transfer compared to Lys-Mt and Glu-Mt. This situation
was supported with cyclic voltammograms that were obtained
3
ꢀ/4ꢀ
with these electrodes for 5.0 mM [Fe(CN) ]
5
at a scan rate of
6
ꢀ
1
0 mV s (Fig. 6a and Table 1). As can be seen from the gure
and the table, Gly-Mt has the narrowest DE values with a smaller
formal potential a value demonstrating again the fastest elec-
tron transfer among all electrode types. Following these nd-
ings, the effect of scanning rate on CV voltammograms was
examined by using Gly-Mt modied GCE (Fig. 6b). The CV
measurements were carried out with varying scan rate between
ꢀ
1
5
.0–1000 mV s and potential range of ꢀ0.4–0.8 V. As a result,
it was observed that developed system has diffusion controlled
behavior between 5.0–1000 mV s scan rates with an equation
ꢀ
1
2
of y ¼ 2.4708x + 10.588, R ¼ 0.984 for Ianodic and equation of
2
y ¼ ꢀ2.493x ꢀ 11.95 R ¼ 0.965 for Icathodic. The range of scan
rate is obviously wider than other Mt modied electrode
ꢀ
1
ꢀ1 12,3
surfaces as mentioned 5.0–200 mV s and 5.0–100 mV s
.
3
.3 Optimization and analytical characterization of A.A-Mt
Fig. 4 SEM images of (a) Mt, (b) Glu-Mt, (c) Lys-Mt and (d) Gly-Mt with
biosensors
1
0 000ꢃ magnification.
Initially, the pH of the medium was optimized via sodium
acetate buffer (between pH 3.5–5.5) and citrate buffer (pH 2.5–
separated particles were also observed. These changes in the
morphologies indicate that the intercalation was accompanied.
3.2 Electrochemical characterization
In order to investigate the surface characterization and electron
transfer mechanisms of different clay mineral surfaces, CV and
Electrochemical Impedance Spectroscopy (EIS) techniques were
utilized. As it is well known, EIS is an effective method for
monitoring electrode surfaces aer modication. Fig.
5
demonstrates the Nyquistic diagrams of bare GCE, amino acid
3
ꢀ/4ꢀ
modied and pristine Mt for 5.0 mM [Fe(CN)
6
]
. As can be
seen from the Fig. 5, Gly-Mt clay mineral lm modied GCE has
the smallest semicircle diameter meaning has the fastest
Fig. 6 (a) Cyclic voltammograms of pristine and modified Mts [in 5.0
3
ꢀ/4ꢀ
ꢀ1
Fig. 5 Nyquistic plots of EIS for bare GCE and amino acid modified Mt mM [Fe(CN)
6
]
at a scan rate of 50 mV s ]. (b) Cyclic voltam-
3ꢀ/4ꢀ
electrodes in the presence of 5.0 mM [Fe(CN)
6
]
and 0.1 M KCl in mograms of Gly-Mt at increasing scan rates [inset graph shows the
5
0 mM sodium phosphate buffer (pH 6.5) at 0.2 V between 0.1 Hz to correlation between the current and square root of the scan rates (5;
ꢀ
1
1
00 kHz.
10; 25; 50; 100; 250; 500; 1000 mV s )].
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Table
electrodes
1
Summary of cyclic voltammetric data for modified
a
o
Epa (V)
Epc (V)
DE (V)
E (V)
Gly-Mt
GCE
Lys-Mt
Glu-Mt
Mt
0.425
0.475
0.450
0.450
0.487
0.225
0.175
0.200
0.200
0.125
0.200
0.300
0.250
0.250
0.362
0.100
0.150
0.125
0.125
0.181
a
o
E ¼ (Epa + Epc)/2.
3.0). Optimum pHs of different A.A-Mt/GOx biolms were found
as 3.5 for Lys-Mt and 4.0 for Gly-Mt and Glu-Mt, respectively
Fig. 7). Aerwards, calibration curves for the each biosensor
(
were plotted at their optimum pHs. It is also known that the pH
optimum of GOx as a free enzyme is 5.5 and in the previous
work, when alkyl-amine intercalated Mts was used for the
construction of GOx biosensor, optimum pH of the system was
Fig. 8 Effect of amino acid modifiers within calibration curves [at ꢀ0.7
V and ambient conditions, 50 mM, sodium-acetate buffer for Gly, Glu-
Mt/GOx and sodium citrate buffer for Lys-Mt/GOx].
12
shied to pH 4.0.
Aer determining the buffer conditions for each biosensing
platform, calibration curves were formed via Gly, Lys and Glu-
Mt. As shown in Fig. 8 higher current values were obtained
from Gly-Mt/GOx sensor with a 30 s response time. The inset
graph demonstrates the current response of the successive
addition of 0.5 mM glucose. Some important analytical
Glu-Mt/GOx) are lower than Gly-Mt/GOx electrode. Further-
more, a biolm prepared with pristine Mt and plain biolm
generated from GA and BSA had been carried out during the
study. However, stability and repeatability of these biolms
were not good. The detection range of the constructed biosen-
sors is lower than other clay–GOx biosensors fabricated with
3
+
parameters for Gly-Mt/GOx are 0.1–1.0 mM as linear range, y
ou Ru(NH )
¼
impregnated Mt structure (detection range: 1.0–15
3
6
2
app
¼
70.573x + 5.148 (R ¼ 0.990) as a linear equation and K
m
34
mM). Furthermore, another GOx sensor based on ruth-
enium(II) complexes of thiosemicarbozones was reported and
0
.7 mM (Michaelis–Menten constant) Imax ¼ 107.8 nA as kinetic
parameters calculated from Lineveawer–Burk diagram for
immobilized enzyme. Additionally, although Lys and Glu-Mt
has wider detection range, their slopes (y ¼ 48.716x + 6.974,
35
its linear range was 0.05–0.5 mM. Additionally, amine-
functionalized naphthalene containing conducting polymer
36
based GOx biosensor was constructed by Azak et al. and its
2
2
R ¼ 0.996 for Lys-Mt/GOx and y ¼ 38.78x + 9.112 R ¼ 0.999 for
linearity was between 0.05 and 1.0 mM which is similar to the
Gly-Mt/GOx biolm and dimethylamine intercalated Mt/GOx
12
biosensor.
Moreover, operational stability and repeatability were also
investigated for Gly-Mt/GOx electrode. In the operational
stability, 65 consecutive trials were performed by adding to
reaction cell 0.5 mM glucose and a 53.8% decrease was observed
in current responses aer 9 h. For the repeatability, aer 10
successive measurements using 0.5 mM glucose, the standard
deviation (S.D) and variation coefficient (c.v) were calculated as
0.45 ꢂ 0.01 mM and 3.98%, respectively.
3.4 Sample application
At the end of the study, sample application was accomplished
for glucose analysis in some beverages such as coke, cherry juice
and zzy. Samples were directly applied to fabricate biosensor
without any pre-treatment. The glucose contents were calcu-
lated using the signals from the linear equation for Gly-Mt/GOx
and shown in Table 2. To compare the results, a spectropho-
tometric method was carried out with the same samples using a
commercial glucose assay kit that includes Trinder reagent.
Each data was given as the mean of 3 measurements ꢂ S.D.
Fig. 7 Effect of pH on the response of clay mineral-based GOx
biosensors [in 50 mM sodium acetate buffer; at the ambient condi-
tions. Electrode composition: GOx (0.5 mg), GA (2.5 mL, 5.0% in pH 7.0,
ꢀ1
sodium phosphate buffer) and BSA (2.5 mL, 1.0 mg mL in pH 7.0
ꢀ1
sodium phosphate buffer) clay mineral (5.0 mL, 1.0 mg mL , in pH 7.0,
sodium phosphate buffer)].
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Table 2 Glucose content of samples obtained by the Gly-Mt/GOx biosensor and reference method
Sample
Gly-Mt/GOx (M ꢂ S.D)
Trinder reagent (M ꢂ S.D)
Recovery %
Coke
Cherry juice
Fizzy
0.055 ꢂ 0.002
0.091 ꢂ 0.007
0.081 ꢂ 0.005
0.059 ꢂ 0.007
0.098 ꢂ 0.003
0.086 ꢂ 0.009
93.2
92.9
94.2
Recovery values indicate that the proposed biosensor can be 11 K. Khezri, V. Haddadi-Asl, H. Roghani-Mamaqani and
used in glucose analysis of real samples.
M. Salami-Kalajahi, J. Polym. Res., 2012, 19, 9868.
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1
4
Conclusion
1
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which are used widely for immobilizing biomolecules. Clay
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37
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