Enzyme immobilization in biosensor constructions: Self-assembled monolayers of calixarenes containing thiols

Article abstract of DOI:10.1039/c3ra47642a

Herein, an amperometric glucose oxidase (GOx) biosensor is presented using calixarenes as an immobilization matrix of the biomolecule. Firstly, thiol-containing calixarenes (Calix-SH) were synthesized, then self-assembled monolayers (SAMs) of Calix-SH on a gold surface were formed and hydroxyl groups of Calix-SH were activated using 1,1′-carbonyldiimidazole (CDI) chemistry. To test the usability of Calix-SH modified surfaces as a biosensor, glucose oxidase was used as a model biological component. After optimization of preparation and working conditions, our results indicate that the Calix-SH/GOx biosensor has a linear range in the range 0.1-1.0 mM (LOD: 0.015 mM) for glucose with a 25 s response time. Finally, the application of the biosensor was examined to detect glucose in real samples. The glucose amounts were calculated as 19.460 ± 0.521 and 31.647 ± 2.125 mM in coke and fizzy drink (with orange), respectively. To confirm the reliability of the Calix-SH/GOx biosensor, the calculated glucose concentrations which were analyzed by the Calix-SH/GOx biosensor were compared to conventional spectrophotometric glucose kits. The glucose amounts in coke and fizzy drink were calculated as 18.509 ± 0.732 mM and 31.579 ± 4.466 mM, respectively.

Full text of DOI:10.1039/c3ra47642a

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PAPER
Enzyme immobilization in biosensor constructions:
self-assembled monolayers of calixarenes
containing thiols
Cite this: RSC Adv., 2014, 4, 19900
a
b
Dilek Odaci Demirkol, Huseyin Bekir Yildiz, Serkan Sayınc and Mustafa Yilmazc
*
*
Herein, an amperometric glucose oxidase (GOx) biosensor is presented using calixarenes as an
immobilization matrix of the biomolecule. Firstly, thiol-containing calixarenes (Calix-SH) were
synthesized, then self-assembled monolayers (SAMs) of Calix-SH on a gold surface were formed and
hydroxyl groups of Calix-SH were activated using 1,10-carbonyldiimidazole (CDI) chemistry. To test the
usability of Calix-SH modified surfaces as a biosensor, glucose oxidase was used as a model biological
component. After optimization of preparation and working conditions, our results indicate that the Calix-
SH/GOx biosensor has a linear range in the range 0.1–1.0 mM (LOD: 0.015 mM) for glucose with a 25 s
response time. Finally, the application of the biosensor was examined to detect glucose in real samples.
The glucose amounts were calculated as 19.460 ꢀ 0.521 and 31.647 ꢀ 2.125 mM in coke and fizzy drink
(with orange), respectively. To confirm the reliability of the Calix-SH/GOx biosensor, the calculated
glucose concentrations which were analyzed by the Calix-SH/GOx biosensor were compared to
conventional spectrophotometric glucose kits. The glucose amounts in coke and fizzy drink were
calculated as 18.509 ꢀ 0.732 mM and 31.579 ꢀ 4.466 mM, respectively.
Received 14th December 2013
Accepted 22nd April 2014
DOI: 10.1039/c3ra47642a
www.rsc.org/advances
platinum, palladium or copper, to name few. Because it allows
controlling rmly dimension and properties of the surface as
Introduction
well as the nature of thiolic ends by various chemistries, SAM
seems a meaningful approach to fabricate stable and repro-
ducible surfaces for biosensor applications.10–12
Immobilized enzymes are widely employed in various biomed-
ical, environmental and industrial applications such as
biosensors, fermentation technology, the food industry, bio-
diesel production and bioremediation, etc.1,2 To fabricate
innovative biosensors and other biomolecule-based diagnostic
techniques, immobilization of the biological component to
solid surfaces is crucial. However, the relevant literature oen
cites that immobilization of enzymes generally causes a decline
in enzyme activity or impaired catalytic characteristics. There-
fore, the choice of efficient immobilization technique is of
paramount signicance to preserve catalytic properties of the
enzyme. To date, different techniques in conjunction with
various immobilization materials such as entrapment in
natural and synthetic polymers,3–6 covalent attachment,7
adsorbtion,8 and cross-linking9 have been of choice to fabricate
enzyme biosensors. Of them, self-assembled monolayers
(SAMs) on the gold surfaces is one of the favorable strategies to
prepare well-ordered structures onto the supports given that
thiols can interact very strongly with metals such as gold, silver,
Calixarenes, cyclic oligomers of phenolic units linked
through the ortho positions, are one of the remarkable macro-
cyclic host molecules.13 They are traditionally prepared via
oligomerization of phenol and formaldehyde.14 Nowadays, they
gain attention in various eld of applications as they are
effortlessly functionalized at either the upper and/or lower rim
of the molecular structure and they form reversible complex
with a large library of compounds including such as ions,
amino acids, peptides and other biomolecules.15,16 By using the
features of capturing cationic molecules, molecular recognition
of aminoacids by calixarenes has opened a gain the application
of them in protein microarrays.17 In another study, Chen et al.
have reported the use of calixarenes for the rst time as enzyme
immobilization material. This group has reported the synthesis
a novel derivative of calixarene, p-tert-butylthiacalix[4]arene
tetra-amine (TC4TA), which was subsequently employed to
adsorb GOx on the TC4TA modied electrode. In this design of
TC4TA/GOD biosensor to detect glucose, the adsorption
mechanism was purely based on ionic interactions between
amino-groups of TC4TA and carboxyl groups of GOx.18 However,
these ionic bonds are notoriously not of high stability as their
strength strictly depends on pH. In this manuscript, we report
the rst synthesis of a thiol-containing calixarene named as
aEge University Faculty of Science Biochemistry Department, 35100 Bornova-Izmir,
Turkey. E-mail: dilek.odaci@ege.edu.tr; Fax: +90 232 311 5485; Tel: +90 232 3115487
bKaramanoglu Mehmetbey University, Kamil Ozdag Science Faculty, Chemistry
¨
Department, 70100, Karaman, Turkey. E-mail: yildizhb@kmu.edu.tr; Fax: +90
3382262116; Tel: +90 3382263840
cSelcuk University Chemistry Department, 42031 Konya, Turkey
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5,11,17,23-tetra-tert-butyl-25,27-bis(3-thiol-1-oxypropane)-26,28- and diminished noise of amperometric measurements and also
dihydroxycalix[4]arene, (SH-Calix, for short hereaer) and its inference effect of some molecules in real samples. Hence,
utilization to modify gold electrode surfaces via creation of self- glutaraldehyde as a crosslinking agent serves multiple purposes
assembled monolayers. Then, we have used GOx as a model with the use of suitable reaction time, pH value and tempera-
enzyme to test application of calixarenes as an immobilization ture as to crosslink enzyme molecules and enzyme to immobi-
material in biosensor constructions. Glucose oxidase (GOx, lization support through their amine groups.28 Besides, the
glucose 1-oxidase, b-D-glucose: oxygen 1-oxidoreductase, EC choice of glutaraldehyde to form covalent bonds eliminates an
1.1.3.4) contains two identical subunits, which are avine additional reduction step to further stabilize the reaction
adenine dinucleotide (FAD) cofactor bound to the polypeptide product.26 In overall, our design of Calix-SH/GOx biosensor was
chains and is a glycoprotein with 74% protein, 16.4% neutral schematized in Scheme 1. Upon the optimization of enzyme
sugar and 2.4% amino sugar.19 At neutral pH values, multipoint loading and pH on the biosensor response, analytical charac-
covalent attachment between multimeric enzymes such as GOx teristics were studied and the application of Calix-SH/GOx
and immobilization material produce stabile enzymes without biosensors was tested to determine the glucose amount in real
subunit dissociation. Because of reduction any conformational samples.
change involved in enzyme inactivation and increasing the
enzyme stability, multipoint covalent attachment provides the
Materials and methods
Materials
rigidication of the enzyme structure.20 Generally, the reactive
amines of aminoacids in the protein surface is useful to bind
them to the surface of support.2
Glucose oxidase [from Aspergillus niger, lyophilized powder,
Type X-S, 147 900 units per g solid; it includes catalase #5 units
per mg protein (foreign activity) and may contain traces of
amylase, maltase, glycogenase, invertase, and galactose oxidase.
Its' protein composition is 65–85% and molecular weight: 160
kDa gel ltration], glucose, 1,10-carbonyldiimidazole (CDI),
dimethyl sulfoxide (DMSO), glutaraldehyde, bovine serum
albumin (BSA) were purchased from Sigma.
In this study, a two step-process was applied to immobilize
Gox on the surface of gold electrode. In the rst step, gold
electrode surface was modied with Calix-SH to acquire a well-
ordered surface to immobilize the enzyme. In here, we should
note that interactions between gold and thiol groups of calix-
arenes direct free hydroxyl groups towards exterior side.
Subsequently, these hydroxyl groups of calixarene are activated
with CDI, upon which the activated support irreversibly reacts
through free amine groups of GOx.21,22 The covalent bonds
between hydroxyl groups of calixarenes well-ordered on the
electrode surface and amine groups of enzyme were formed
with 1,10-carbonyldiimidazole (CDI) chemistry.23,24 Of course,
the prime purpose of this technique is to bring the monolayer of
GOx molecules on the surface of the calixarene-modied elec-
trode, leaving all enzyme molecules equally accessible.25 Next,
we have added glutaraldehyde to form the cross-links between
BSA and GOx. However, glutaraldehyde did not solely cross-link
BSA and GOx, but also cross-links BSA enzymes and BSA-to-GOx
enzymes, leading to a fabrication of a biomembrane.26 Natu-
For the synthesis of calixarenes, TLC analyses were carried
out on DC Alufolien Kieselgel 60 F254 (Merck). Generally,
solvents were dried by storing them over molecular sieves
˚
(Aldrich; 4 A, 8–12 mesh). All reactions, unless otherwise noted,
were conducted under nitrogen atmosphere. All starting mate-
rials and reagents used were of standard analytical grade from
Merck or Aldrich used without further purication. All aqueous
solutions were prepared with deionized water that was passed
through a Millipore milli-Q Plus water purication system.
Apparatus
rally, acquiring a biomembrane works in our favor as it offers PalmSens electrochemical measurement unit (Palm Instru-
several advantageous such as saturation of any non-specic ments, Houten, Netherlands) was used to carry out chro-
binding sites,27 superior stability of Calix-SH/GOx biosensors noamperometric measurements. The experiments were
performed in a reaction cell (10 mL) at room temperature using
of three electrodes conguration, consisting of gold working
electrode (BASI, USA), Ag/AgCl reference electrode (3 M KCl,
Metrohm, Switzerland) and a platinum counter electrode
(Metrohm, Switzerland). Pharmacia LKB Novaspec II spectro-
photometer (LKB Biochrom, England) was used in colorimetric
experiments as a reference method in sample applications.
Melting points were determined on a Gallenkamp apparatus
1
in a sealed capillary glass tube and are uncorrected. H NMR
spectra were recorded on a Varian 400 MHz spectrometer.
Elemental analyses were performed using a Leco CHNS-932
analyzer.
For the surface characterization of the prepared calixarene-
based biosensors, JEOL5600-LU model scanning electron
microscope (SEM) was used. To obtain samples for SEM analy-
Scheme 1 Schematic representation of Calix-SH/GOx biosensor.
sis, gold slides were modied with same procedure which was
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used to prepare Calix-SH/GOx biosensors. The images were phosphate buffer and spread over the Calix-SH modied gold
taken via using an acceleration voltage of 20 kV.
The spectrophotometric data to calculate enzyme activity 4 C overnight. Finally, the mixture of 2.5 mL of 1.0% of glutar-
and protein amount have been collected by means of a Lambda aldehyde and 2.5 mL of BSA (0.5 mg mLꢂ1) solutions prepared in
electrodes. Aer that, the electrodes were allowed to stand at
ꢁ
35 UV/vis spectrometer purchased from Perkin-Elmer (USA).
pH 7.0; 50 mM phosphate buffer was spread over the surface of
the modied electrodes and dried for 1 h at room temperature.
Before and aer immobilization steps, GOx activity of the
prepared biosensors was checked according to ABTS method by
enzymatically determining the concentration of the produced
hydrogen peroxide by means of peroxidase (POD), according to
the following procedure: 1 mL of ABTS (0.003 M), 0.3 mL of POD
(6 U), 1 mL of glucose (0.3 M), and 0.7 mL of potassium phos-
phate buffer (50 mM; pH 6.0) were mixed and incubated for 5
min at 30 ꢁC. Then electrodes were immerged into the reaction
mixture and absorbance was followed for 3 min at 420 nm. The
concentration of ABTS was in excess in respect to that of H2O2
so that the reaction was limited by the concentration of the
latter product. H2O2 concentration was calculated by using the
extinction coefficient of ABTS (3420 ¼ 43 200 Mꢂ1 cmꢂ1). One
unit (U) of enzyme activity was dened as the amount of enzyme
that catalyzed the production of 1 mmol H2O2 per minute under
the experimental conditions.30,31 Protein contents of GOx solu-
tion before and aer immobilization procedure were calculated
according to the Bradford assay.32
Synthesis of SH-Calix, 5,11,17,23-tetra-tert-butyl-25,27-bis(3-
thiol-1-oxypropane)-26,28-dihydroxycalix[4]arene
The compounds 1 and 2 were synthesized by procedures pub-
lished in the literature.13,29 SH-Calix named as 5,11,17,23-tetra-
tert-butyl-25,27-bis(3-thiol-1-oxypropane)-26,28-dihydroxycalix-
[4]arene (3) is herein reported for the rst time (Scheme 2). For
the synthesis of Calix-SH, a mixture of dialkyl bromide of p-tert-
butylcalix[4]arene (2) (0.5 g, 0.561 mmol) and thiourea (0.14 g,
1.88 mmol) in CH3CN (40 mL) was reuxed. The reaction was
monitored by using a TLC (CH2Cl2–hexane(1 : 1)). Aer 45 h,
the solvent was removed under reduced pressure. The remain-
ing was mixed with 0.19 g of KOH (3.423 mmol) and aliquot of
21 mL of deionized water and allowed to reux for 2 h. The
residue was extracted with 1 M HCl and CHCl3, dried over
MgSO4 to afford pure compound 3 with 40% yield-m.p.: 203–
205 ꢁC. 1H NMR (400 MHz CDCl3): d 1.19 (s, 18H, but), 1.22
(s, 18H, but), 1.57–1.69 (m, 2H, –SH), 2.42–2.48 (m, 4H, –CH2–),
3.24 (t, 4H, J ¼ 8.0 Hz, –CH2–S), 3.38 (d, 4H, J ¼ 12.8 Hz, Ar–
CH2–Ar), 4.01 (t, 4H, J ¼ 4.8 Hz, –CH2–O), 4.23 (d, 4H, J ¼ 12.8
Hz, Ar–CH2–Ar), 7.01 (s, 4H, ArH), 7.07 (s, 4H, ArH), 9.13 (s, 2H,
–OH). Anal. calcd. For C50H68O4S2: C, 75.33; H, 8.60. Found (%);
C, 75.45; H, 8.79.
Measurement procedure
All electrochemical measurements were performed at room
temperature in an open vessel lled with the vigorously stirred
10 mL of 50 mM acetate buffer solution, pH 4.5. Increasing
concentrations of glucose were added denite volumes of the
stock solution of 1.0 M glucose (in pH 4.5, 50 mM acetate
buffer). The response of the Calix-SH/GOx biosensor shows the
decrease of dissolved O2 content upon exposure to glucose
solutions. According to the reaction scheme, GOx uses b-D-
glucose as substrate and converts it to D-glucono-1,5 lactone and
hydrogen peroxide by oxidizing glucose at carbon-1:33
Fabrication of Calix-SH/GOx biosensors
Before each experiment, gold electrodes were polished with
alumina powder (Gamma, 0.05; 0.1; 0.3; 1.0 mm). Then it etched
in 0.5 M H2SO4 solution by cyclic-potential scanning between
0 and 1.5 V until a reproducible voltammetric response was
obtained. Initially, 1 mg Calix-SH and 50 mg CDI were dissolved
in 0.5 mL of DMSO and the gold electrode was immersed in the
mixture of Calix-SH and CDI for 1.5 h at room temperature. Then
1 mg GOx was dissolved in 5 mL of pH 8.2; 0.1 M sodium
b-D-glucose + O2 / D-glucono-1,5-lactone + H2O2
In rst generation amperometric biosensors, oxygen
consumption and the formed hydrogen peroxide can be fol-
lowed at ꢂ0.7 and +0.7 V, respectively. It is known that most
common metabolites such as uric acid and ascorbic acid get
oxidized and interfere with the electrochemical signal at higher
potentials. It is therefore essential to apply the lowest possible
electrode potential.10,33 According to this data, The decrease of
dissolved O2 concentration was determined as the analytical
signal of the biosensor at ꢂ0.7 V in this study.34 The steady-state
current was typically achieved in 25 s. Aer each measurement,
the working buffer was changed and the electrodes were washed
with distilled water.
Scheme 2 The synthetic route for synthesis of 5,11,17,23-tetra-tert-
butyl-25,27-bis(3-thiol-1-oxypropane)-26,28-dihydroxycalix[4]arene
(3). Reaction conditions: (i) K2CO3, CH3CN, NaI, 1,3-dibromopropane;
(ii) thiourea, CH3CN, KOH.
Sample application
The developed biosensors were tested with real samples (coke
and zzy with orange). Samples were degassed and diluted with
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working buffer and then injected into the reaction buffer their functionality. In our study, thiol containing calixarenes
instead of glucose. Calibration curves were used to determine (SH-Calix) are synthesized and gold electrode surfaces were
the glucose contents in measured samples. The samples were modied by them via construction of SAMs. And free hydroxyl
also applied to a commercial enzyme assay kit based on spec- groups of SH-Calix were functional point to immobilize GOx in
trophotometric Trinder reaction (Cromatest, Glucose MR, Cat. targeted position using CDI chemistry. To increase signal/noise
no. 1129010) as the reference method and results were ratios and to prevent the negative effects of interfering
compared with those obtained with the constructed biosensors. substances in samples, the surface was covered with BSA. Aer
In the Trinder reaction, the glucose is oxidized to D-gluconate by fabrication of Calix-SH/GOx biosensor, initially the obtained
glucose oxidase (GOx) with the formation of hydrogen peroxide. biomembrane was characterized. To evaluate of the conduc-
In the presence of peroxidase (POD), a mixture of phenol and tivity of the biomolecule-modied gold electrode, Calix-SH/GOx
4-aminoantipyrine (4-AAP) is oxidized by hydrogen peroxide to biosensor was characterized by cyclic voltammetry (CV) using
form a red quinoneimine dye proportional to the glucose Fe(CN)63ꢂ as an electrochemical probe. CV obtained at the bare,
concentration in the sample.35
SH-Calix and SH-Calix/GOx modied Au electrodes immersed in
aqueous 0.1 M KCl containing 5 mM Fe(CN)6 is shown in
3ꢂ
Fig. 2A, respectively. A reversible electrochemical response for
Results and discussion
Characterization of SH-Calix
p-tert-Butylcalix[4]arene (1) and its dialkyl bromide derivative (2)
were synthesized according to the literature routes.29,36 The
substitution of dibromide derivative of p-tert-butylcalix[4]arene
(2) was conducted in the presence of CH3CN with thiourea and
KOH solution to afford the cone conformer dithio substituted
p-tert-butylcalix[4]arene (3) in 40% yield. The 1HNMR spectra of
(3) has a typical AX pattern for the methylene bridge proton
(ArCH2Ar) of calixarene by appearing two doublets at 3.38 and
4.23 ppm (12.8 Hz)37 (Fig. 1).
Application of SH-Calix on the fabrication of enzyme
biosensors
Calixarenes are synthesized by the oligomerisation of phenol
and formaldehyde, offer several advantages in synthesis and
applications as supramolecular platforms for molecular recog-
nition, sensing and self-assembly, catalysis, nanotechnology,
and drug discovery.14,15 In enzyme-based detection technolo-
gies, immobilization of proteins on the solid surface, without
losing their activity is the critical point. Calixarenes are useful
macromolecules to keep their structural motifs and thereby
Fig. 2 (A) Cyclic voltammetric characterization of bare, Calix-SH and
Calix-SH/GOx modified electrodes conducted in acetate buffer (pH
4.5) containing 5 mM Fe(CN)63ꢂ. (B) CV response of Calix-SH/GOx
Fig. 1 1H-NMR spectrum of 5,11,17,23-tetra-tert-butyl-25,27-bis(3- biosensor at different scan rates. (C) CV of Calix-SH/GOx biosensor in
thiol-1-oxypropane)-26,28-dihydroxycalix[4]arene (3).
the absence and presence of glucose.
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3ꢂ
Fe(CN)6 was obtained. Well-dened oxidation and reduction
Effect of pH on the biosensor response
peaks are observed at about 0.258 and 0.165 V for bare electrode
(peak-to-peak separation of 93 mV), 0.244 and 0.151 V for SH-
Calix modied electrode (peak-to-peak separation of 93 mV)
and 0.338 and 0.139 V for Calix-SH/GOx-modied Au electrodes
(peak-to-peak separation of 199 mV). According to obtained
graphs, there is a decrease in the peak current when the Au
electrode was coated step-by-step. Fig. 2B displays CVs of SH-
Calix/GOx modied Au electrodes at the different scan rates (5;
10; 25; 50; 75; 100; 125; 150; 175; 200 mV sꢂ1). The peak current
increased linearly with the increasing square root of scan rate
potential (v1/2). This suggests that the reactions on the SH-Calix/
GOx modied Au electrodes were reversible and the mass
transport phenomenon is mainly diffusion controlled. Fig. 2C
depicts the change of the cyclic voltammograms of Calix-SH/
GOx biosensor in the presence of glucose.
The working pH is an important factor that can signicantly
inuence the performances of the biological catalysts. To obtain
optimum pH of Calix-SH/GOx biosensor, the pHs were tested
ranges from 3.5 to 5.5 (50 mM acetate buffers). The pH effect on
the response of the Calix-SH/GOx biosensor is shown in Fig. 4.
Maximum amperometric response was achieved at pH 4.5. Free
glucose oxidase has a broad activity range of pH 4–7 (ref. 38) (as
quoted by the manufacturer, Sigma-Aldrich) and obtained pH
value as an optimum pH is agreement with the needed pH
values reported for the free enzyme.
Effect of enzyme amount on the biosensor response
To decide the enzyme content to obtain maximum sensitivity,
the Calix-SH/GOx biosensors were fabricated by various GOx
amounts (0.5 mg; 1.0 mg and 2.0 mg) and calibrated for glucose
detection (Fig. 5). The highest signal appeared in the use of 1.0
mg of the enzyme. An enzyme loading of 0.5 mg was insufficient
for detection of glucose and the efficiency of biosensor which
was prepared using 2.0 mg GOx was lower than that for 1.0 mg.
As mentioned before, protein content and GOx activity of the
prepared Calix-SH/GOx biosensor were checked using Bradford
and ABTS method, respectively. Protein contents of Calix-SH/
GOx biosensors which were fabricated using 0.5; 1.0 and 2.0 mg
enzyme were calculated as 0.135; 0.30 and 0.64 mg according to
the Bradford assay, respectively. Enzyme activities for Calix-SH/
GOx biosensors which were fabricated using 0.5; 1.0 and 2.0 mg
enzyme were found as 3.50; 24.56 and 11.72 units according to
ABTS method, respectively. Because of the difficulties in the
diffusion of glucose and oxygen in thick surface which contain
biocomponents, higher amounts of GOx caused a decrease in
the current. Linearity for substrates and the currents which
were obtained using various electrodes modied with different
enzyme amounts are depends on the electrode species, immo-
bilization technique and also enzyme.3,4,33,39,40 Furthermore, the
purity of the enzyme can bring about decrease in activity of
enzyme when using an excess of protein.41 Here, GOx includes
The morphologies of SH-Calix and SH-Calix/GOx modied
Au electrodes were characterized by SEM and the obtained
images could be seen in Fig. 3. Data, was obtained from SEM
images supported CV results. Aer immobilization steps and
covering with BSA, the surface was smooth and the spaces
between ridges were disappeared.
Fig. 4 Effect of pH on the biosensor response of Calix-SH/GOx
Fig. 3 SEM images of Calix-SH films (A) and Calix-SH/GOx films on the biosensor (in sodium acetate buffer, 50 mM, ꢂ0.7 V; error bars show
gold electrode surface.
S.D. of three measurements).
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Fig. 5 Effect of enzyme amount on the biosensor response (in sodium
acetate buffer, 50 mM, pH 4.5, ꢂ0.7 V; error bars show S.D. of two or
three measurements).
Fig. 6 Calibration curve for glucose (in sodium acetate buffer, 50 mM,
pH 4.5, ꢂ0.7 V; error bars show S.D. of three measurements. Inset:
time dependent current response with the addition of 0.25 mM
glucose).
catalase, amylase, maltase, glycogenase, invertase, and galac-
tose oxidase. During immobilization, contaminant proteins
also was immobilized. When GOx was increased, the thickness
of biocomponent layer was increased excessively because of
impurities. Hence in all the following experiments, the enzyme
amount of biosensor was kept at 1.0 mg. When 1.0 mg enzyme
was used to prepare Calix-SH/GOx biosensor, activity yield was
87%. The yield was calculated considering the specic activity
values for free and immobilized enzyme, which are expressed as
U per mg protein.
response time was faster than the biosensor which was
prepared using BSA, the sensor response was decreased aer
ve measurements for a standard glucose solution.
The repeatability of measurement was obtained by consec-
utive seven trials of 0.25 mM glucose using the same electrode.
The relative standard deviation (R.S.D.) was calculated to be
4.7%. To evaluate the electrode-to-electrode reproducibility,
three electrodes were prepared under the same conditions in
different days. The result showed a R.S.D. of 2.6%, indicating a
satisfactory reproducibility. To test operational stability, the
biosensors response to 0.5 mM glucose standard solutions was
Analytical characteristics
Different types of alkanethiols have been investigated as an followed. Aer measurements in 3 h, no decrease was observed
immobilization matrix to fabricate biosensors via forming self in biosensor response. In order to compare the analytical
assembled monolayers (SAMs). In this study, thiol-containing performance of Calix-SH/GOx biosensors with the glucose
calixarene structures were tested as an alternative immobiliza- biosensors based on calixarene in the literature, the character-
tion support or coatings to alkanethiols for the preparation of istics of them were shown in Table 1. Pandya et al. developed
biosensors. Because of its effectiveness in bioconjugation, non-enzymatic sensor combined with spectroscopic techniques
covalent bonds were used to combine hydroxyl groups of cal- for the detection of glucose based on calix[4]arene/phenyl
ixarene and amine groups of enzyme. The electrochemical boronic acid functionalized gold nanoparticles.44 Due to its
signal of the Calix-SH/GOx biosensors toward glucose was ability to reversibly bind diol-containing compounds, its selec-
investigated by chronoamperometrically. As shown in Fig. 6, tivity is low. But enzymatic sensors show the affinity and
current change was proportional to the glucose concentration selectivity to their substrates. Coupling of enzymes and trans-
in the range from 0.1 to 1.0 mM, and the linear regression ducer are affiliated to success of immobilization techniques.
equation was I (nA) ¼ 159.732 + 31.624 (mM) (R2 ¼ 0.995) with a Various types of materials such as synthetic/natural polymer
detection limit of 0.015 mM (S/N ¼ 3) and 25 s response time membranes, composites and nanomaterials have been used to
(inset of Fig. 6 shows the signal aer glucose addition). When fabricate biological material based detection systems. Calixar-
the concentration of glucose was more than 1.0 mM, substrate enes can be used as an alternative to these materials for the
saturation was observed, showing a typical Michaelis–Menten immobilization of biological component of biosensors. For this
kinetic mechanism.
aim, Chen et al. used the calixarenes for the rst time as an
Some proteins such as gelatin, albumin have been added to enzyme immobilization matrix and aer immobilization of
architectures which has been used to prepare biosensor glucose oxidase, amperometric glucose detection was carried
immobilization matrix.42,43 In the absence of these macromol- out.18 Firstly, p-tert-butylthiacalix[4]arene tetra-amine (TC4TA)
ecules, the repeated measurements are not carried out because was synthesized and spread on the surface of the platinum disk
of matrix decomposition containing biological material. To electrode. Then, using EDC/NHS coupling reaction glucose
show importance of BSA in immobilization matrix, glucose oxidase was immobilized on the TC4TA modied electrode.
biosensor was fabricated without BSA and glutaraldehyde. The Finally, amperometric response to glucose was followed as
obtained biosensor signal was noisy. Although the biosensor an amperometrically at +0.60 V vs. SCE. In our study,
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Table
Paper
1
Comparison of different calixarene-based glucose sensing techniques to Calix-SH/GOx biosensor according to analytical
characteristics
Support
Immobilization technique
Principle of detection
Linearity
LOD
Ref.
—
Non-enzymatic
UV-visible spectrophotometry
Amperometric
5–100 nM
0.08–10 mM
4.3 nM
—
44
18
Platinum
disk electrode
Gold electrode
GOX immobilization to adsorbed calixarene
via EDC/NHS coupling reaction
GOX immobilization to SAMs calixarene
via CDI coupling reaction
Amperometric
0.1–1.0 mM
0.015 mM
This work
5,11,17,23-tetra-tert-butyl-25,27-bis(3-thiol-1-oxypropane)-26,28-
dihydroxycalix[4]arene (SH-Calix) was synthesized for the rst
time and used to modify gold electrode surfaces via creation of
self-assembled monolayers (SAMs). Then glucose oxidase was
immobilized the calixarene-modied surface. The success of
the prepared biosensor depends on the immobilization tech-
nique. Among of other immobilization procedures, SAMs have
some advantages such as easy preparation of well-designed
functional surface, simplicity of preparation, versatility,
stability, reproducibility and the possibility of introducing
different chemical functionalities.
Effect of some interferents on the biosensor response
Fig. 7 Interference by various compounds on the Calix-SH/GOx
biosensor used in glucose determination (in sodium acetate buffer, 50
mM, pH 4.5, ꢂ0.7 V; error bars show S.D. of three measurements.
[Glucose]: 0.25 mM; cysteine [Cys]: 0.1 mM; phenol [Ph]: 0.1 mM;
ethanol [EtOH]: 0.1 mM).
The inference studies of Calix-SH/GOx biosensors have been
investigated in the presence of some compounds such as
galactose, mannose, cysteine, phenol and ethanol. Cysteine and
phenol were chosen as a model compound of thiolic and
phenolic electroactive species, respectively. Ethanol was used to
test the interference effect of this compound to biosensor
response when Calix-SH/GOx biosensor is applied to analyze Calix-SH/GOx biosensors and the reference method (n: 3;
glucose in alcoholic beverages. Galactose and mannose recovery%: 100%), respectively. The results proposed that Calix-
(0.1 mM) were selected to show substrate specicity of GOx and SH/GOx biosensor can be successfully used for the determina-
added to reaction medium instead of glucose, but no response tion of glucose in beverages.
were found for these monosaccharide. 0.1 mM of cysteine,
phenol and ethanol were added to reaction buffer in the pres-
ence of 0.25 mM glucose and the signal was followed chro-
Conclusions
noamperometrically (Fig. 7). No signicant change in the
current response to glucose was obtained in the presence of
oping a calixarene-based biosensor for monitoring glucose.
these compounds.
In summary, this study was dened the probability of devel-
Firstly, thiol containing calixarenes were synthesized. Then the
electrochemical biosensing platform was established by SAMs
Sample application
of Calix-SH and GOx was immobilized the calixarene-modied
gold surfaces via CDI chemistry. Aer that, the stability of
immobilized enzyme was increased using BSA and glutaralde-
hyde and also addition of them prevents the interference of the
other molecules in sample matrix. The current change was
followed based on oxygen consumption during enzymatic
reaction at ꢂ0.7 V. Aer optimization and characterization
studies, the proposed Calix-SH/GOx biosensor was used
successfully for glucose detection in real samples and it could
provide as a valuable tool for other elds to determine glucose.
The glucose concentrations of coke and zzy with orange were
quantitatively analyzed using Calix-SH/GOx biosensor under
optimized conditions. Samples were added to reaction buffer as
a substrate instead of glucose and the concentration of glucose
in samples were calculated using calibration graph. The results
were then compared to calculated glucose concentrations which
were analyzed by spectrophotometric glucose kits as a conven-
tional method in order to conrm the reliability of Calix-SH/
GOx biosensor. The glucose amount of coke was calculated as
19.460 ꢀ 0.521 and 18.509 ꢀ 0.732 mM using Calix-SH/GOx
biosensors and the reference method (recovery%: 105%),
respectively (n ¼ 3, data are given as the mean ꢀ S.D. (standard
Acknowledgements
deviation)). And glucose amount of zzy (with orange) was Authors would like to thank the European Union through the
calculated as 31.647 ꢀ 2.125 and 31.579 ꢀ 4.466 mM using COST Action CM1202 “Supramolecular photocatalytic water
19906 | RSC Adv., 2014, 4, 19900–19907
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splitting (PERSPECT-H2O)” and the Scientic and Technolog- 21 H. M. Lee, S. O. Song, S. H. Cha, S. B. Wee, K. Bischoff,
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113T022) for the nancial support of this research.
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