Calixarene modified montmorillonite: A novel design for biosensing applications

Article abstract of DOI:10.1039/c4ra11818a

Here we report the synthesis, characterization and application of calixarene (Calix) modified montmorillonite (Mt) as a platform for bio-applications such as biomolecule immobilization and biosensing technologies. This modification enhanced the biomolecule immobilization capability of Mt. Initially, amino-functionalised calixarenes (Calix-NH₂) were synthesized and used as a modifier. X-ray diffraction, Fourier transform infrared spectroscopy, zeta potential and thermal gravimetric analysis were performed to verify the modification of the clay minerals. For the biosensor construction, Calix-NH₂ modified Mt (Calix-NH₂/Mt), bovine serum albumin (BSA), glutaraldehyde (GA) and pyranose oxidase were immobilized on the surface of a glassy carbon electrode which was then referred to as a Calix-NH₂/Mt/PyOx biosensor. After optimization of the enzyme amount and pH, analytical characteristics were investigated in detail.

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ARTICLE
Calixarene Modified Montmorillonite: A Novel
Design for Biosensing Applications
Cite this: DOI: 10.1039/x0xx00000x
a
b
c
a
Burak Sonmez, Serkan Sayin, Esra Evrim Yalcinkaya, Didem Ag Seleci, Huseyin Bekir
b
a,d*
a,d*
Yildiz, Dilek Odaci Demirkol, and Suna Timur
Received 00th January 2012,
Accepted 00th January 2012
Here we report the synthesis, characterization and application of calixarene (Calix) modified
montmorillonite (Mt) as platform for the bioꢀapplications such as biomolecule
immobilization and biosensing technologies. This modification enhanced the biomolecule
immobilization capability of Mt. Initially, aminoꢀfunctional calixarenes (CalixꢀNH ) were
a
DOI: 10.1039/x0xx00000x
www.rsc.org/
2
synthesized and used as a modifier. Xꢀray diffraction, Fourier transform infrared spectroscopy,
zeta potential and thermal gravimetric analysis were performed to verify the modification of
clay minerals. For the biosensor construction, CalixꢀNH modified Mt (CalixꢀNH /Mt), bovine
2
2
serum albumin (BSA), glutaraldehyde (GA) and pyranose oxidase were immobilized on the
surface of glassy carbon electrode which is then called as CalixꢀNH /Mt/PyOx biosensor. After
2
optimization of the enzyme amount and pH, analytical characteristics were investigated in
detail.
Introduction
bonding with guest molecules, calixarenes can selectively bind
20ꢀ22
different inorganic and organic compounds.
Chen et al. reported
Immobilization of the enzymes in a suitable matrix is crucial for
fabrication of biosensor systems and other enzymeꢀbased diagnostic
techniques. In the literature, various techniques and immobilization
the synthesis of pꢀtertꢀbutylthiacalix[4]arene tetraꢀamine and
demonstrated the use of calixarenes as enzyme immobilization
23
material. Recently, 5,11,17,23ꢀtetraꢀtertꢀbutylꢀ25,27ꢀbis(3ꢀthiolꢀ1ꢀ
oxypropane)ꢀ26,28ꢀdihydroxycalix[4]arene (SHꢀCalix) was
1ꢀ5
materials have been attempted to fabricate enzyme biosensors.
Clays are one of the advantageous materials to design different
immobilization matrices for biomolecule immobilization due to their
high stability, good adsorptive capacity, large specific surface area,
synthesized and used to modify gold electrode via formation of selfꢀ
assembled monolayers. Then, glucose oxidase was immobilized on
24
the SHꢀCalixꢀmodified surface.
6
stick out adhesiveness and low costs. Laponite, montmorillonite
(
Mt), nontronite and layered doublehydroxides (LDHs) are well
This work composed of three parts as follows; (i) the synthesis and
characterization of 5,17ꢀdiaminoꢀ25,27ꢀbis[Nꢀ(2ꢀ
known Smectite clay minerals that already were exploited in
numerous biosensor studies.
phyllosilicate and composed of silica tetrahedral sheets layered
between alumina octahedral sheets. To date it has been utilized in
different biotechnological and biomedical applications.
instance, Joshi et al. used Mt as drug carrier for timolol maleate and
demonstrated the intercalation of timolol maleate into the interlayer
of Mt at different pH. Folic acid modified Mt was used as a cell
specific adhesion material for HeLa cells. Demir et al were
successfully applied trimethylamine modified Mt to design microbial
biosensor systems. Besides, glucose oxidase and laccase were also,
immobilized on dimethylamine and histidine modified Mt matrices
7
ꢀ11
Mt is a naturally occurring cationic
aminoethyl)]calix[4]azacrown(CalixꢀNH ), (ii) modification of Mt
2
with CalixꢀNH salt which is then, used as a platform for the enzyme
2
immobilization, (iii) biosensor (CalixꢀNH /Mt/PyOx) applications.
2
12ꢀ14
For
Pyranose oxidase (PyOx) was selected as a model enzyme.
Glutaraldehyde (GA) and bovine serum albumin (BSA) were used to
test the specificity of the sensor. Amine groups of Calixꢀ NH played
2
1
5
an important role in the stabile immobilization of the enzyme by
crosslinking with GA. CalixꢀNH BSA, GA and PyOx were mixed
1
6
2,
and immobilized onto Glassy carbon electrode (GCE). The
17
25
enzymatic reaction of PyOx is given below;
9
, 18
for glucose and phenol sensing, respectively.
DꢀGlucose + O2
2ꢀdehydroꢀDꢀglucose + H O2
2
Calixarenes which are cyclic oligomers, are synthesized traditionally
via oligomerization of phenol and formaldehyde. Due to their
excellent ability to form host–guest complexes and their multi center
The consumption of the oxygen due to the enzymatic activity was
related to the substrate concentration. The response signals were
followed at ꢀ0.7 V vs Ag/AgCl. After optimization studies, Calixꢀ
1
9
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NH /Mt/PyOx biosensor was calibrated for glucose and applied for and then, filtered and evaporated to dryness. The crude was
2
o
the glucose analysis in a real sample.
recrystallized from acetone. Yield: %95, m.p.; 269ꢀ272 C.
ꢀ
1
1
FTIR (ATR): 1660 cm (C=O). H NMR (400 MHz DMSO):
2.69 (t, 3H, J= 4.4 Hz, ꢀ NH ), 2.92ꢀ2.95 (m, 2H, ꢀCH ꢀ), 3.16ꢀ
3 2
δ
+
Materials and Methods
3
.45 (m, 10H, ꢀCH2ꢀ over shielded by the solvent), 3.63ꢀ3.74
Materials. Pyranose oxidase (EC 1.1.3.10, from Coriolus sp., ≥ 2.7
Units/mg), GA solution (25%, v/v), bovine serum albumin (BSA)
glucose and all other chemicals were ordered from Sigma Aldrich.
Mt was supplied by Southern Clay Products, Inc. (CEC = 92
meq/100 g). Thin layer chromatography (TLC) analyses were carried
out on DC Alufolien Kieselgel 60 F254 (Merck). The solvents were
dried by storing them over molecular sieves (Aldrich; 4 Å, 8ꢀ12
mesh). All reactions, unless otherwise noted, were conducted under
nitrogen atmosphere. All starting materials and reagents used were
of standard analytical grade from Merck or Aldrich and used without
further purification. All aqueous solutions were prepared with
deionized water that was passed through a Millipore MilliꢀQ Plus
water purification system.
(
m, 4H, ArꢀCH ꢀAr), 3.98ꢀ4.04 (m, 8H, ArꢀCH ꢀAr and Arꢀ
2
2
NH ), 4.52ꢀ4.59 (m, 4H, OꢀCH ꢀ), 6.89 (t, 2H, J= 7.6 Hz,
ArH), 7.21 (d, 4H, J= 7.6 Hz, ArH), 7.57ꢀ7.74 (m, 2H, ꢀOH),
2
2
13
7
.99ꢀ8.23 (m, 6H, ArH and ꢀNH).
DMSO): 31.07 (Arꢀ H ꢀAr), 37.26 (NHꢀ
H ), 53.04 (Nꢀ H ), 54.81 (Nꢀ
C
NMR (100 MHz
δ
C
C
H ), 38.98 (NH ꢀ
2
2
2
C
C
C
H ), 74.20 (Oꢀ
2
C
H ), 124.96
2
2
2
(
(
Arꢀ
Arꢀ
C
), 126.02 (Arꢀ
C
), 129.46 (Arꢀ
), 152.99 (ArOꢀ
C=O). Anal. Calcd. For C H N O : C, 66.94; H, 6.65;
C
), 130.10 (Arꢀ
C
), 133.56
C), 144.60 (NH ꢀArꢀ
C
C), 153.60 (ArOꢀ
C),
2
1
69.26 (
3
8
45
6
6
N, 12.33. Found (%); C, 67.01; H, 6.73; N, 12.28.
Synthesis of Calix-NH2. pꢀtertꢀButylcalix[4]arene
calix[4]arene ( ), 1,3ꢀOꢀdimethyl ester calix[4]arene derivative
and the compound Nꢀ(2ꢀaminoethyl)calix[4]azacrown were
synthesized according to literature procedures.
compounds 5,17ꢀdinitroꢀ25,27ꢀbis[Nꢀ
2aminoethyl)]calix[4]azacrown and 5,17ꢀdiaminoꢀ25,27ꢀ
(1),
2
3
4
2
6ꢀ29
The
5
(
6
bis[Nꢀ(2ꢀaminoethyl)]calix[4]azacrown are herein reported for
the first time.
Synthesis of compound 4. Yield; 70.5%, m.p.; 250ꢀ252
o
ꢀ1
1
C.FTIR (ATR): 1678 cm (C=O). H NMR (400 MHz
CDCl3): 2.13ꢀ2.33 (m, 2H, ꢀNH ), 2.53 (t, 2H, J=5.6 Hz, ꢀ
CH ꢀN), 2.74ꢀ2.81 (m, 6H, ꢀCH ꢀ), 3.46 (d, 4H, J= 13.6 Hz,
δ
2
2
2
ArꢀCH ꢀAr), 3.53ꢀ3.56 (m, 4H, ꢀCH ꢀ), 4.21 (d, 4H, J= 13.6
2
2
Hz, ArꢀCH ꢀAr), 4.55 (s, 4H, OꢀCH ꢀ), 6.73ꢀ6.79 (m, 4H, ArH),
2
2
6
(
.85 (d, 4H, J=7.6 Hz, ArH), 7.12 (d, 4H, J=7.6 Hz, ArH), 7.19
s, 2H, ꢀOH), 8.32 (t, 2H, J=5.6 Hz, ꢀNH).Anal. Calcd. For
C H N O : C, 70.13; H, 6.51; N, 8.61. Found (%); C, 70.05;
3
8
42
4
6
H, 6.63; N, 8.55.
Synthesis of compound 5. HNO (65%, 20 mL) was added to
3
the solution of
4 (0.7 g, 1.076 mmol) in 20 mL of CH Cl . The
2 2
mixture was stirred at room temperature for 1 h. Then, 50 mL
water was poured into the mixture, and then, filtered. The solid
was washed with water to neutralize and then, dried. The crude
was recrystallized from acetone. Yield; 56.5%, m.p.; 208ꢀ
Scheme 1. The synthetic route for compound 6. Reaction conditions: (i)
Phenol, AlCl ; (ii) K CO , CH CN, methylbromoacetate; (iii) Tris (2ꢀ
3
2
3
3
3 2 2
aminoethyl)amine, MeOH/toluene; (iv) HNO , CH Cl ; (v) RaneyꢀNi,
hydrazine, MeOH.
o
ꢀ1
1
2
10 C.FTIR (ATR): 1660 cm (C=O). H NMR (400 MHz
+
Preparation of Calix-NH /Mt. The Mt was organically modified by
2
DMSO):
H, ꢀCH2ꢀover shielded by the solvent), 3.69ꢀ3.73 (m, 8H, ꢀ
CH ꢀ and ArꢀCH ꢀAr), 4.19ꢀ4.30 (m, 4H, ArꢀCH ꢀAr), 4.58
δ 2.60ꢀ2.70 (m, 5H, ꢀCH ꢀ and ꢀ NH ), 3.15ꢀ3.47 (m,
2 3
a cationꢀexchange reaction between Mt and CalixꢀNH . The Calixꢀ
2
6
NH , as an intercalating agent, was firstly protonated with 1.0 M
2
2
2
2
HCl aqueous solutions to adjust the pH to 2.0–3.0. The required
(
brs, 4H, OꢀCH ꢀ), 6.89 (t, 2H, J=7.6 Hz, ArH), 7.21 (d, 4H,
2
amount (two time equivalent of the CEC) of the CalixꢀNH salt was
2
J=7.6 Hz, ArH), 7.63 (brs, 2H, ꢀOH), 8.18ꢀ8.24 (m, 6H,ArH
o
dissolved in deionized water at approximately 50 C. 1.0 g of Mt was
and ꢀNH). Anal. Calcd. For C H N O : C, 61.53; H, 5.57; N,
3
8
41
6
10
preliminarily dispersed in deionized water by using a mechanical
1
1.33. Found (%); C, 61.44; H, 5.81; N, 11.38.
stirrer. The CalixꢀNH solution was added to the dispersion of the
2
clay mineral particles, and the mixture was exchanged for 24 h at
room temperature using vigorous stirring. The resulted organoꢀclay
Synthesis of compound 6. Raney Ni (1.0 g) was added to the
solution of (0.35 g, 0.473 mmol) and hydrazine monohydrate
2.8 mL) in MeOH. The reaction mixture was refluxed for 10 h.
The reaction temperature was then cooled to room temperature,
5
mineral (CalixꢀNH /Mt) was collected by filtration and repeatedly
2
(
washed with hot deionized water until no chloride ions were detected
2
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ART18ICLE
upon adding 0.1 M aqueous AgNO . It was dried in a vacuum oven the substrate concentration were followed at –0. 7 V vs Ag/AgCl.
3
o
at 50 C for 2 days to a constant weight.
The consumption of molecular oxygen in the enzymatic reaction was
monitored at given potential. PyOx oxidizes glucose to 2ꢀdehydroꢀ
Dꢀglucose and the signals were correlated indirectly to the substrate
Characterization of Calix-NH /Mt. For the characterization,
initially, modified and pristine samples were examined by Xꢀray
diffraction (XRD) Spectrometers (Philips E'xpert Pro; CuꢀKα
2
9
,17,18,24
concentrations consumed during the enzymatic reaction.
radiation, λ=1.54056 Ǻ) to determine the interlayer spaces of Mt and After each measurement, the electrodes were washed with distilled
CalixꢀNH /Mt. The samples were scanned under the diffraction water and the working buffer was refreshed.
2
−1
angle 2θin the range of 4.0–70.0 with a scanning rate of 2.0 min .
The interlayer distance was determined by the diffraction peak, using
the Bragg equation (nλ=2dsinθ). Fourier transform infrared (FTIR)
spectra of clay minerals were recorded using a Perkin Elmer Pyris 1
FTIR Spectrometer with KBr pellet. Thermal degradation was
studied with a thermogravimetric analyzer (Perkin Elmer Pyris 1
TGA/DTA) by heating the samples from ambient temperature to
Sample application. CalixꢀNH /Mt/PyOx biosensor was used to
2
analyze the glucose concentration in coke and fizzy samples. The
sample was also analyzed with Trinder reagent which is a
commercial enzyme assay kit based on spectrophotometric analysis.
This assay includes oxidation of glucose to Dꢀgluconate catalyzed by
glucose oxidase with the formation of hydrogen peroxide. In the
presence of peroxidase, a mixture of phenol and 4ꢀaminoantipyrine
is oxidized by hydrogen peroxide to form a red quinineimine dye
ꢀ
1
1
000 °C at 10 °C min under a 10 bar dry air atmosphere. The
electrokinetic properties of clay minerals were determined by
measuring the zetaꢀpotential of particles with a ZetaꢀMeter 3.0+
3
1
proportional to the glucose concentration in the sample. The
glucose concentration in the real sample was measured by direct
addition of samples into working buffer, instead of glucose. The
amount of glucose in the samples was calculated from corresponding
calibration curves obtained with the standard glucose solutions.
(with a Zeiss DR microscope, GTꢀ2 type quartz cell, molybdenumꢀ
cylinder anode, and platinumꢀrod cathode electrode). For this
purpose, the clay mineral samples were stirred overnight in the
deionized water to obtain well dispersed particles. The zeta potential
of the Mt dispersions was estimated from the measured
30
electrophoretic mobility by employing the Smoluchowski equation.
Results
The value of the zeta potential assigned to the dispersions was the
average of the data obtained from at least 10 experiments. All of the
samples were dried in a vacuum oven before the analyses at 50 C
Synthesis of a calixarene derivative substituted at the both
rims with primary amine groups. pꢀtertꢀButylcalix[4]arene
1
o
was chosen as the starting material, and it was transformed to
for 2 days.
the dealkylated derivative
according to known procedures (see Scheme 1).
of the diester derivative
presence of MeOH/toluene led to produce compound
2
and then the diester derivative
3
2
6ꢀ28
Treatment
Scanning Electron Microscopy (SEM) images were registered by
using SEM (JEOL5600ꢀLU). The images were taken via using an
acceleration voltage of 20 kV
3
with tris(2ꢀaminoethyl)amine in the
4
2
9
according to the literature procedure. Then, a mixture of
compound with 65% HNO in DCM was reacted at room
temperature for 1 h yielded the
Upon reduction of nitro groups with RaneyꢀNi, the target
compound , which was functionalized at the both rims of
4
Calix-NH /Mt biosensor. Glassy carbon electrodes (GCE) were
3
2
p
ꢀnitro substituted derivative 5.
polished with 0.5 mm alumina slurry. To remove alumina residues
on the electrode, ultrasonic cleaning for about 2–3 min in 1:1 ethanol
distilled water was carried out. Afterwards, 1.0 mg PyOx (in 2.5 µL,
sodium phosphate buffer pH 7.0, which equals to 2.7 Units), 5.0 µL
6
Calix with the primary amine groups, was synthesized in 95%
yield (see Scheme 1).
of CalixꢀNH /Mt solution (1.0 mg/mL in sodium phosphate buffer
2
The characterization of the synthesized Calix derivatives was
performed by FTIR, H and C NMR spectroscopy and given
pH 7.0), 2.5 µL of BSA (1.0 mg/ mL in sodium phosphate buffer,
pH: 7.0) and 2.5 µL of glutaraldehyde (5.0%, in sodium phosphate
1
13
buffer, pH 7.0) were mixed. Finally, 12.5 µL of this mixture was as supplementary data (Fig.S1ꢀ Fig. S5).The FTIR spectra of
dropped on the electrode surface and allowed to dry at room
signify that their characteristic peaks(C=O) appear at 1678
temperature for 1 h. The same procedure was used to prepare the Mt cm for
modified PyOx biosensors in which only pristine Mt was used NMR spectrum of
instead of CalixꢀNH modified clay mineral.
aminoethyl) aiming at
J= 5.6 Hz, ꢀCH2ꢀN), 2.74ꢀ2.81 (m, 6H, ꢀCH2ꢀ), 3.53ꢀ3.56 (m,
Electrochemical measurements. Amperometric measurements 4H, ꢀCH2ꢀ) and 8.32 ppm (t, 2H, J= 5.6 Hz, ꢀN
) placed on the
4ꢀ
6
ꢀ
1
ꢀ1
1
4
and 1660 cm for both
, the additional peaks of Tris (2ꢀ
2.13ꢀ2.33 (m, 2H, ꢀN ), 2.53 (t, 2H,
5 and 6 (Fig. S1). In the H
4
δ
H
2
2
H
performed using a Palm Sens Potentiostat (Palm Instruments, spectrum. Thus, these peaks confirm the structure of
4 (Fig.
1
Houten, Netherlands). All experiments were carried out in a reaction S2). H NMR spectrums of
5
which are nitro derivative of
by disappearing of two
.0) at room temperature using of three electrode configuration. protons of aromatic at para positions on the spectrum (Fig.
4
cell in working buffer (10 mL sodium phosphate buffer, 50 mM, pH clearly verifies the structure of
5
6
GCE as a working electrode, Ag/AgCl as a reference electrode (3.0
M KCl, Metrohm, Switzerland) and Pt as a counter electrode
S3).Obtained compound
protons at 3.98 ppm belonging ArꢀNH groups in the HꢀNMR
6 by reduction of nitro groups has four
1
2
1
3
(Metrohm, Switzerland) were used. Initially, the biosensors were
spectrum (Fig. S4). In addition, in the C NMR spectra of
can be seen the peak belonging to the =O groups appears at
69.26 ppm, (Fig. S5).
6, it
equilibrated in the working buffer in the reaction cell and then,
glucose was added. The response signals which were proportional to
C
1
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Characterization of Calix-NH /Mt. Each layer of natural Mt and the exchange resulted in an increase in the basal spacing.
2
consists of octahedrally coordinated cations (typically Mg, Al The basal spacing of CalixꢀNH /Mt is increased to 13.06 Å
2
and Fe) sandwiched by tetrahedrally coordinated cations (2
θ
= 6.76). The interlayer space height, ꢁ
as 1.66 Å by subtracting the average layer thickness of Mt (11.4
leads to a net negative surface charge that is compensated by an Å) from that of CalixꢀNH /Mt. It is attributed that the cation
d, has been calculated
4+
3+
(
typically Si and Al). Isomorphous substitution of Si by Al
2
2
+
2+
2+
interlayer of exchangeable, hydrated cations (Ca , Mg , Cu , exchange process achieved between the interlayer cations of Mt
+
+
Na , H ). The surface of Mt is hydrophilic and this makes and calixarene salt intercalated into the Mt layers.
difficult the interaction of the clay mineral with the enzyme
substrate. Therefore, it is necessary to make the surface of Mt
less hydrophilic to compatible with a chosen enzyme. Various
methods have been proposed for making clay minerals more
3
2
1
1
200
000
hydrophobic. In our case, the methodology is based on the
replacement of the exchangeable hydrated cations with
organophilic cations. The clay minerals were characterized by
FTIR, XRD, TGA/DTG and zeta potential measurements. The
1
200
8
6
00
00
1000
400
200
0
8
6
4
00
00
00
FTIR spectra of pristine Mt and CalixꢀNH /Mt are presented in
Fig. 1. In the FTIR spectrum of Mt (Fig. 1 A), the band at 3630
is ascribed the ꢀOH bond corresponding to the
characteristic stretching for AlꢀAlꢀOH bond in the octahedral
layer of Mt. The band at around 1630 cm is related with Hꢀ
OH stretching vibration. An intense peak at around 1000 cm
is the characteristic absorption band for Mt corresponding to Siꢀ
O stretching. After modification with CalixꢀNH (Fig. 1B)
A
2
4
5
6
7
8
9
10
−
1
cm
−
1
B
−
1
200
0
2
0
10
20
30
40
50
60
70
characteristic absorption bands for both Mt and modifier were
observed. In addition to Mt band, characteristic peaks (C=O) of
CalixꢀNH were shifted towards to 1730 cm . Also, vibration
2θ
ꢀ
1
2
2
Fig.2 XRD patterns of Mt (A) and CalixꢀNH /Mt (B).
−
1
−
1
peaks at 2980 cm and 2850 cm for
–
CH stretching and at
CH2 bending are observed, supporting the
intercalation of CalixꢀNH salt between the silicate layers.
−
1
1
360cm for –
2
The thermogravimetric (TG) and derivative thermogravimetric
DTG) curves for pristine Mt and CalixꢀNH /Mt are shown in Fig. 3
(
2
and Fig. 4, respectively. The TGA thermogram of pristine Mt reveals
two major temperature regions of mass loss, one below 200 °C and
another above 500 °C. The first step observed from room
temperature is attributed to the dehydration of physically adsorbed
water and dehydration of water molecules adsorbed around metal
cations on exchangeable sites in Mt. The last step observed in the
temperature region of 500ꢀ700 °C is attributed to the
dehydroxylation of Mt. After the exchange with the CalixꢀNH salt,
2
thermal decomposition of the CalixꢀNH /Mt takes place in three
2
regions. In the first region, below 150 °C, takes place the evolution
of adsorbed water. The second region occurs between 400ꢀ500 °C
due to the decomposition of incorporated organic molecules, and the
last one over the temperature range of 600–700 °C which is ascribed
to the dehydroxylation of the structural OH units of the organoclay
2
Fig.1 FTIR spectra of Mt (A) and CalixꢀNH /Mt (B).
mineral. From Fig. 4, it is observed that the CalixꢀNH /Mt presents
2
The Xꢀray diffraction (XRD) technique is frequently used to lower thermal stability than the pristine Mt.
detect the basal spacing between the interlayer of the clay
Also, the TGA results were used to examine the amount of organic
mineral particles. XRD pattern of pristine Mt and Calixꢀ
component. The organic content of the CalixꢀNH /Mt was calculated
NH /Mt is shown in Fig. 2 Pristine Mt yields characteristic
2
2
from the residue of the CalixꢀNH /Mt after being burnt for over 30
min at 800 °C, using the following equations:
diffraction peaks at 2
to the basal spacing d001(11.4 Å). From the XRD pattern of
θ
= 7.74. This diffraction peak corresponds
2
33
CalixꢀNH /Mt (Fig. 2), it can be seen a diffraction peak
appeared at the lower angles of the diffractogram indicated the
2
ꢆꢇꢈꢉꢊꢋꢌꢍꢉꢎꢏꢂꢐꢑꢑ°ꢒ
%
ꢀ
ꢁꢂꢃ%ꢄ ꢅ
(1)
ꢓ.ꢔꢕꢖ
intercalation of CalixꢀNH molecules in the interlayer spaces of
2
Mt. The ions of the clay mineral were exchanged with
ammonium cations of calixarene to prepare the organoclays,
ꢗꢘꢙꢂꢃ%ꢄ ꢅ 100% ꢚ ꢀꢁ%
(2)
4
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The factor of 0.987 in the first equation corrects for loss of zeta potentials of the samples were measured as ꢀ42.0±2.24 and
physically adsorbed water of the pristine Mt, being in this case 1.3% ꢀ29.70±0.63 mV for Mt and CalixꢀNH /Mt, respectively. After
2
mass loss as taken from TGA thermogram. From these equations,
the amount of organic component is calculated as 16.67%.
each modification step, the zeta potentials shifted to lessꢀ
negative values as a result of adsorption of positively charged
calixarene salt at the surface or interlayer of the mineral.
Optimization studies. Effect of pH and the modifier on the
100
biosensor response were investigated for CalixꢀNH /Mt/PyOx
2
biosensor. The effect of pH on the response of the biosensor
was studied between pH 5.5 and 7.0 in sodium acetate and
sodium phosphate buffer (50 mM) by using glucose (0.25 mM).
The lower biosensor response obtained with lower or higher
pHs than 6.0. Therefore, pH 6.0 is selected as the optimum for
9
9
8
8
5
0
5
0
A
B
CalixꢀNH /Mt/PyOx biosensor (Fig. 5). Previously, the
2
optimum pH was obtained at 10.5 for the PyOx biosensor in
which the enzyme was immobilized in the Osmium redox
2
5
polymer. In another work PyOx immobilized in carbon
nanotube modified carbon paste electrode and optimum pH was
39
reported as 7.5. It is known that the optimum pH of free PyOx
0
200
400
T
600
800
4
0
is 7.0. In our work, optimum working pH of PyOx was shifted
to an acidic pH due to the presence of positive groups in the
(o
C)
structure of CalixꢀNH intercalated Mt. It is clear that optimum
pH is varied depending on the matrices used for the
biomolecule immobilization.
2
Fig.3 TG thermograms of Mt (A) and CalixꢀNH
2
/Mt (B).
0
,00
A
B
-
-
-
-
-
0,01
0,02
0,03
0,04
0,05
1
00
8
6
4
0
0
0
0
200
400
600
800
20
0
T
(o
C)
Fig.4 DTG thermograms of Mt (A) and CalixꢀNH
2
/Mt (B).
5.0
5.5
6.0
pH
6.5
7.0
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
Fig. 5 Effect of pH on the response of CalixꢀNH
2
/Mt/PyOx
Furthermore, the amount of immobilized enzyme affects
41,42
biosensor sensitivity directly.
To observe this effect, three
different CalixꢀNH /Mt biosensors prepared with 0.5 mg, 1.0
2
3
4
determination 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
transport of particles or blood cells, sensors and biosensors,
and 2.0 mg of PyOx and the amounts of other components were
kept constant. Fig. 6 demonstrates that lower enzyme amounts
(
0.5 mg) cause lower responses due to inadequate enzyme
activity. When enzyme amounts were increased to 1.0 mg the
highest signal responses were obtained. Furthermore, if 2.0 mg
of enzyme was immobilized on the electrode, bioactive layer
becomes thicker and this causes decreasing of signals.
3
5ꢀ38
membrane efficiency and microfluidics.
In this study, the
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measured three times with a 20 s response time. The calibration
curve of CalixꢀNH /Mt/PyOx biosensor was defined by the equation
2
2
of y = 3.16x + 0.02 (R = 0.997) and demonstrated in Fig.8. Data
were given as mean ± S.D. The limit of detection (LOD) was also
calculated as 0.50 µM using S/N=3. Various clay mineral based
0.5 mg Enzyme
1.0 mg Enzyme
2.0 mg Enzyme
2
1
1
0
0
.0
.5
.0
.5
.0
4
3,44
glucose biosensors using laponite,
alginate/layered double
45
hydroxides, dimethyl and trimethyl modified Mt were reported
17,18
previously.
When CalixꢀNH /Mt/PyOx is compared with
2
18
theearlier works, a lower LOD has been observed. The comparison
of various PyOx biosensors reported in literature was also given in
the supplementary data as Table S1.
Moreover, repeatability and operational stability of the biosensor
were investigated and 10 succeeding measurements were performed
using 0.1 mM glucose. The S.Dand variation coefficient (c.v) were
calculated as 0.1±0.001 mM and 1.06%, respectively. The
reproducibility of the biosensor was also evaluated using 4 different
enzyme electrodes prepared at different times, using 0.1 mM
glucose. The relative standard deviation (RSD) was found as 0.05%.
0.0
0.5
1.0
1.5
2.0
[
Glc](mM)
Fig. 6 Effect of enzyme amount on the biosensor response (in sodium
phosphate buffer, 50 mM, pH 6.0, at ꢀ 0.7 V; error bars show standard
deviation (S.D) of three measurements).
For the operational stability of CalixꢀNH /Mt/PyOx biosensor, 80
2
measurements during 72 h were carried out and after these
measurements only 30% decrease was observed. Besides, SEM
Both types of clay mineral samples, pristine and CalixꢀNH modified
2
Mt were applied to examine the effect of CalixꢀNH modification on
2
images, obtained for Mt (Fig. 9 A) and CalixꢀNH /Mt (Fig. 9 B),
2
the biosensor response. The amperometric responses of the Mt/PyOx
proved the decoration of Mt with CalixꢀNH . The changes in the
2
and CalixꢀNH /Mt/PyOx biosensors were linear in the range 0.01ꢀ0.5
2
surface morphologies depending on the modifier types were shown
in Fig. 9 C and Figure 9 D, respectively. According to these images,
it can be said that the surfaces of Mt/PyOx biosensors were different
2
mM glucose by the equations of y= 2.33x + 0.03 (R = 0.99) and y =
2
3
.92x + 0.07 (R = 0.99) respectively. Fig. 7 shows that Calixꢀ
NH /Mt/PyOx biosensor has higher current values as well as higher
2
from CalixꢀNH /Mt/PyOx biosensors. In the presence of
2
sensitivity (that can be seen from the slope of the linear graph) in
compared to Mt/PyOx.
glutaraldehyde as a crossꢀlinker, covalent bonds between the free
amine groups of CalixꢀNH2, PyOx and BSA were formed and the
surface morphology of CalixꢀNH /Mt/PyOx biosensors was
2
differentiated from Mt/PyOx biosensors.
1.5
1.0
0.5
0.0
y=3.11x+0.02
2
R =0.998
0
.0
ꢀ0.5
ꢀ1.0
ꢀ1.5
ꢀ2.0
ꢀ2.5
ꢀ3.0
ꢀ3.5
Glc
0
100
200
300
t (s)
0.0
0.1
0.2
0.3
0.4
0.5
[Glc] (mM)
Fig. 7 Effect of the presence of CalixꢀNH
structure on the biosensor response (in sodium phosphate buffer, 50 mM, pH
.0, at ꢀ 0.7 V; error bars show S.D of three measurements).
2
as a modifier in the matrix
6
Fig. 8 Calibration curve for glucose (in sodium phosphate buffer, 50 mM, pH
, ꢀ0.7 V; error bars show S.D of three measurements. Inset: time dependent
current response with the addition of 0.5 mM glucose).
6
Analytical Characterization. CalixꢀNH /Mt/PyOx biosensor was
2
calibrated under optimum conditions by the addition of glucose
standards between 0.01ꢀ0.5 mM. All the response signals were
6
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Table 1. Glucose content of some beverages obtained by the biosensor and
the reference method.
Calix-
Trinder
Reactive
Recovery
%)
NH /Mt/PyOx
Sample
2
(
(
mM±SD)
(mM)
Coke
Fizzy
5.00 ± 0.0
5.04 ± 0.1
6.0 ± 0.15
99.2
96.5
5.79 ± 0.47
Conclusions
In conclusion, a promising material which is a good candidate for
several biotechnological applications was synthesized, characterized
and applied as an enzyme immobilization and biosensor platform.
PyOx was chosen as a model enzyme to fabricate Mt based
biosensors. The CalixꢀNH /Mt/PyOx sensor has shown good
2
linearity with low detection limit. At the final step, the biosensor was
applied for glucose analysis in real samples and the obtained data for
the clay biosensor were in good agreement with reference method.
This system could apply different bioꢀanalysis systems for the
biotechnological applications.
2
Fig. 9 SEM images of Mt (A), CalixꢀNH /Mt (B) with 5000 X magnification,
Mt/PyOx (C), and CalixꢀNH /Mt/PyOx (D) with 10000 X magnification.
2
Scale bars show 1.0 µm for A and B and 300 nm for C and D.
Interferences.Ascorbic acid, uric acid and 3ꢀacetamino phenol (0.1
mM of each compound) were used to evaluate the inference effect
Acknowledgements
The Authors would like to thank the European Union through
the COST Action CM1202 “Supramolecular photocatalytic
water splitting (PERSPECTꢀH2O)” and the Scientific and
Technological Research Council of Turkey (TUBITAK Grant
on the CalixꢀNH /Mt/PyOx biosensor. It is known that at higher
2
potentials, most common metabolites such as uric acid and ascorbic
46
acid get oxidized and interfere with the electrochemical signal.
Therefore, effect of interferents to biosensor signals was evaluated Numbers 113T022) for the financial support of this research.
by using the working potentials of ꢀ0.7 V. The differences in current
levels were compared with that of standard glucose solutions and
were calculated as a relative response value. Except ascorbic acid, no Notes and references
a
change was observed in the current response to glucose in the
presence of interfering compounds.
Ege University Faculty of Science, Biochemistry Department 35100
BornovaꢀIzmir/Turkey.
b
KTO Karatay University, Department of Materials Science and
Sample application. Under optimized conditions the glucose Nanotechnology Engineering, 42020 Konya, Turkey.
c
concentrations of coke and fizzy were detected using Calixꢀ
Ege University Faculty of Science Chemistry Department, 35100,
NH /Mt/PyOx biosensor. The concentration of glucose in the
2
BornovaꢀIzmir/Turkey.
d
samples was calculated using the calibration graph. The results were
compared with calculated glucose concentrations which were
analyzed by reference method (Trinder Reagent) to confirm the
Ege University, Institute of Drug Abuse Toxicology &Pharmaceutical
Sciences, 35100ꢀBornova, Izmir/Turkey.
accuracy of designed biosensor. The calculated glucose amounts of Electronic Supplementary Information (ESI) available: [details of any
both samples are shown in Table 1. According to data the use of the
supplementary information available should be included here]. See
DOI:10.1039/b000000x/
CalixꢀNH /Mt/PyOx biosensor provided very similar results for the
2
kit data (with the recovery of 99.2%).
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