A benzimidazole/benzothiazole-based electrochemical chemosensor for nanomolar detection of guanine

Article abstract of DOI:10.1039/c4ra12892c

The electrochemical detection of guanine was accomplished using benzimidazole/benzothiazole-based imine-linked Co(iii) complexes 2, 4 and 6 with platinum electrodes. Linear sweep voltammetry (LSV), differential pulse voltammetry (DPV) and cyclic voltammet

Full text of DOI:10.1039/c4ra12892c

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DOI: 10.1039/C4RA12892C
Graphical Abstract
A benzimidazole/benzothiazole-based electrochemical chemosensor for nanomolar
detection of guanine
Hemant Sharma, Narinder Singh,* and Doo Ok Jang*
The electrochemical detection of guanine was accomplished using benzimidazole/ benzothiazole-
based imine-linked Co(III) complexes with platinum electrodes.

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A benzimidazole/benzothiazole-based electrochemical ch
D
e
OI
m
: 10.1
o
03
s
9
e
/C
n
4RA
s
12
o
892C
r
for nanomolar detection of guanine
a
a,
b,
Hemant Sharma, Narinder Singh * and Doo Ok Jang *
a
Department of Chemistry, Indian Institute of Technology Ropar (IIT Ropar), Rupnagar, Panjab, India 140001
b
Department of Chemistry, Yonsei University, Wonju 220-710, Republic of Korea
nsingh@iitrpr.ac.in; dojang@yonsei.ac.kr
Abstract: The electrochemical detection of guanine was accomplished using benzimidazole/ benzothiazole-
based imine-linked Co(III) complexes 2, 4 and 6 with platinum electrodes. Linear sweep voltammetry (LSV),
differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were the major analytical techniques used
to explore the recognition behavior of the complexes. The detection limit, linear range of detection and
-1
2
sensitivity for complex 2 (16.6 nM, 3.5-10 µM and 4.01 µA µM cm ), complex 4 (13.4 nM, 5.0-120 µM and
-1
2
-1
2
3
.18 µA µM cm ) and complex 6 (11.3 nM, 2.5-100 µM and 2.0 µA µM cm ) were calculated. Advantages
of this methodology include simplicity, an unmodified electrode, high sensitivity and reproducibility.
Introduction
In recent years, biomolecular recognition has gained considerable attention due to its widespread role in
1
-8
biological, chemical and environmental systems. The fundamentals of biomolecular recognition originate
from the structure of deoxyribonucleic acid (DNA). DNA is a genetic material consisting of two strands, which
are attached to each other through non-covalent interactions between nitrogenous bases (adenine, guanine,
9, 10
cytosine and thymine). These nitrogenous bases bind in a particular way, called complementary base paring.
This complementary binding and non-covalent interaction are the basis for molecular recognition. Furthermore,
nitrogenous bases are important compounds in biological systems because they are involved in several
processes, including energy transduction, the storage of genetic information, metabolic cofactors and cell
1
1, 12
signaling.
The determination and estimation of nitrogenous bases are particular for guanine because it can be
oxidized easily as compared to the other purine bases. Up to the present time, various analytical methods have
been developed for the determination and estimation of nitrogenous bases as achieved by liquid
13-15
chromatography, mass spectroscopy, UV-vis and fluorescence spectroscopy.
Electrochemical techniques
offer several benefits over other detection techniques, including simplicity, relative low cost, portability, high
performance with lower background, sensitivity and applicability to turbid samples. Various efforts regarding
the electrochemical sensing of guanine have been reported, which used modified electrodes with different
1

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1
6
17
18
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electron transfer mediators such as cobalt hexacyanoferrate, cobalt(II) phthalocyanine, redox polymer and
DOI: 10.1039/C4RA12892C
1
9
cobalt oxide nanostructures . However, these techniques have suffered from various limitations including a
high detection limit, reduced stability of mediators and electro-catalysts, and lengthy, time-consuming
preparation methods. We previously reported fluorogenic and chromogenic chemosensors for cations and
20-22
anions using benzimidazole/benzothiazole-based signaling units.
Herein, we wish to report
benzimidazole/benzothiazole-based Co(III) complexes for the direct sensing of guanine without modification of
the surface of electrodes. To the best of our knowledge, this is the first attempt to use
benzimidazole/benzothiazole-based sensors to recognize guanine via electrochemical techniques.
Experimental section
General
Chemicals were purchased from Aldrich and were used without further purification. The NMR spectra were
1
13
recorded on JNM-ECS400 (JEOL) spectrophotometer operated at 400 MHz for H NMR and 100 MHz for C
NMR spectra. The splitting patterns were visualized as s (singlet), bs (broad singlet), d (doublet), dd (doublet of
doublet), t (triplet), and m (multiplet). The electrochemical measurements were recorded on a Potentiostat-
Galvanostat BASI EPSILON. The X-ray diffraction (XRD) pattern was recorded with a PANalytical X’PERT
PRO diffractometer, operated at 45 KV, 40 mA using Ni-filtered Cu- Kα radiations with a scan speed of
1
0º/min for 2θ in a range from 10 to 70.
Recognition studies
o
All recognition studies were performed at 25±1 C, and before recording any spectrum, a sufficient incubation
period with shaking was performed to ensure the uniformity of the solution. For binding studies, a stock
solution of respective receptors was prepared in DMSO/H
2
O or a THF/H
2
O (8:2; v/v) solvent system. For
-
-
-
-
-
-
-
-
binding studies, tetrabutylammonium anion salt (F , Cl , Br , I , NO
3
, H
2
PO
4
3 4
, CH COO and HSO ), sodium or
disodium salt (ATP, ADP, NADH, NAD and NADP), and adenine, cytosine, guanine and uracil was employed.
To perform this experiment, volumetric flasks (5 mL) were taken and each contained an analyte solution along
with 10 µM of respective ligand solution and tetrabutylammonium perchlorate as the supporting electrolyte (0.1
M). Receptors showed selectivity and were titrated with the respective analyte. The titrations were performed in
10-mL volumetric flasks that contained a 10 µM solution of receptor and a different concentration of analyte.
Furthermore, stoichiometry of the complex between the host and guest was revealed by a method of continuous
variation or Job’s plot, and was confirmed from mass spectroscopy. The interference studies were performed in
5
mL volumetric flasks, containing a solution of complex 2, 4 or 6 along with guanine and an equal molar of
particular competing an anion or a biomolecule. These solutions were kept for 1 h and then measurements were
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carried out using desired techniques. Electrochemical detection was performed in a single-compartment cell
DOI: 10.1039/C4RA12892C
under a nitrogen atmosphere at 25 ºC, with a Pt disk working electrode (3 mm diameter), a platinum wire
counter electrode (0.5 mm diameter) and an Ag/AgNO
3
reference electrode. The Pt disk working electrode was
(diameter of 50 nm). It was washed with distilled
abraded to a mirror finish using emery paper and α-Al
2
O
3
water followed by ultrasonic cleaning in ethanol and deionized water. Tetrabutylammonium perchlorate was
used as the supporting electrolyte (0.1 M).
Synthesis of compound 1
A solution of 2-aminobenzimidazole (499 mg, 3.75 mmol) and 2,3-dihydroxybenzaldehyde (600 mg, 4.35
mmol) in methanol (20 mL) was heated to reflux for 10 h. A reddish-brown-colored compound was separated
out when the reaction mixture was cooled to room temperature. After filtration, the solid was washed with
1
methanol three times. This resulted in a 67% yield (640 mg); mp 244-245 °C; H NMR (400 MHz, DMSO-d
6
)
13
δ 12.73 (bs, 1H, -OH), 11.93 (bs, 1H, -OH), 9.56 (s, 1H, -CH=N), 7.55-6.80 (m, 8H, Ar, NH); C NMR (100
MHz, DMSO-d ) δ 166.8, 154.3, 145.0, 146.3, 142.9, 134.5, 123.4, 122.9, 122.6, 120.7, 120.1, 119.9, 119.1,
11.8. Anal. Calcd. for C14 : C, 66.40; H, 4.38; N, 16.59. Found: C, 66.34; H, 4.23; N, 16.71.
6
1
11 3 2
H N O
Synthesis of compound 2
A solution of compound 1 (678 mg, 2.68 mmol) and Co(NO ) (244 mg, 1.34 mmol) in THF (50 mL) was
3 2
stirred at room temperature for 3 h. Tetrabutylammonium nitrate was added to the above solution for aerial
oxidation. After filtration, a dark-brown-colored powder was obtained in 65% yield (980 mg). mp ˃ 275 °C;
+
ESI-MS (m/z): 618.3 [M+1] for [2.(H
2
O)
3
]. Anal. Calcd. for C28
H
26CoN
6
O
7
: C, 54.46; H, 4.24; N, 13.61.
Found: C, 54.61; H, 4.29, N, 13.59.
Synthesis of compound 3
A solution of 2-aminobenzothiazole (499 mg, 3.33 mmol) and salicylaldehyde (499 mg, 4.09 mmol) in
methanol (20 mL) was heated to reflux for 10 h. A yellow-colored product was obtained when the reaction
mixture was cooled to room temperature. After filtration, the product was obtained in 69% yield (583 mg). mp
1
1
35-136 °C; H NMR (400 MHz, DMSO-d
6
) δ 11.48 (s, 1H, -OH), 9.40 (s, 1H, -CH=N), 8.04 (d, J = 7.6 Hz,
1H, Ar), 7.91 (d, J = 8.0 Hz, 2H, Ar), 7.49 (t, J = 7.7 Hz, 2H, Ar), 7.42 – 7.37 (m, 1H, Ar), 7.02–6.94 (m, 2H,
1
3
Ar); C NMR (100 MHz, DMSO-d
6
) δ 171.0, 166.3, 161.1, 151.7, 136.2, 134.5, 131.5, 127.3, 125.9, 123.1,
23.0, 120.4, 120.1, 117.5. Anal. Calcd. for C14 OS: C, 66.12; H, 3.96, N, 11.02. Found: C, 66.37, H,
.85; N, 10.92.
1
3
10 2
H N
3

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DOI: 10.1039/C4RA12892C
Synthesis of compound 4
A solution of compound 3 (676 mg, 2.66 mmol) and Co(NO ) (244 mg, 1.34 mmol) in THF (50 mL) was
3
2
stirred at room temperature for 3 h. Tetrabutylammonium nitrate was added to the above solution for aerial
oxidation. After filtration, a dark-brown-colored product was obtained in 70% yield (1.05 g). mp 180-185 °C;
+
ESI-MS (m/z): 565.0 [M] for 4. Anal. Calcd. for C28
H18CoN
4
O
2
S
2
: C, 59.47; H, 3.21; N, 9.91. Found: C,
59.44; H, 3.15; N, 9.89.
Synthesis of compound 5
A solution of 2-aminobenzothiazole (499 mg, 3.33 mmol) and 2-hydroxy-5-nitrobenzaldehyde (601 mg, 3.6
mmol) in methanol (20 mL) was heated to reflux for 10 h. A yellow-colored product was separated out when
the reaction mixture was cooled to room temperature. After filtration, the product was obtained in 62% yield
1
(
617 mg). mp 220-221 °C; H NMR (400 MHz, DMSO-d
6
) δ 10.26 (s, 1H, -OH), 8.38 (d, J = 2.8 Hz, 1H, -
CH=N), 8.31 (dd, J = 9.1, 2.9 Hz, 1H, Ar), 7.60 (d, J = 7.7 Hz, 1H, Ar), 7.45 (s, 1H, Ar), 7.28 (d, J = 7.9 Hz,
1
3
1
H, Ar), 7.19 – 7.11 (m, 2H, Ar), 6.96 (t, J = 7.5 Hz, 1H, Ar); C NMR (100 MHz, DMSO-d
66.3, 165.0, 153.1, 140.2, 131.3, 126.0, 124.9, 122.7, 121.4, 121.4, 119.0, 118.2. Anal. Calcd. for
S: C, 56.18; H, 3.03; N, 14.04. Found: C, 56.39; H, 3.05; N, 13.99.
6
) δ 189.6, 167.0,
1
14 9 3 3
C H N O
Synthesis of compound 6
A solution of compound 5 (795 mg, 2.66 mmol) and Co(NO ) (244 mg, 1.34 mmol) in THF (50 mL) was
3
2
stirred at room temperature for 3 h. Tetrabutylammonium nitrate was added to the above solution for aerial
oxidation. After filtration, a dark-brown-colored powder was obtained in 60% yield (1.04 g). mp ˃ 275 °C;
+
ESI-MS (m/z): 724.5 [M+1] for [6.(H
2
O)
2
CH
3
OH]. Anal. Calcd. for C29
H
24CoN
6
O
9
S
2
: C, 48.14; H, 3.34; N,
11.61. Found: C, 48.43; H, 3.66; N, 11.71.
Results and Discussion
Synthesis of receptors and cobalt complexes
Benzimidazole/benzothiazole-based imine-linked receptors 1, 3 and 5 were prepared by condensation
reactions. The condensation reactions of 2-aminobenzimidazole or 2-aminobenzothiazole with benzaldehyde
derivatives were performed in methanol (Scheme 1). The products were obtained in good yields and
characterized in agreement with the structures. To make them electrochemically active, cobalt complexes of
receptors 1, 3 and 5 were synthesized and characterized by mass spectroscopy and elemental analysis.
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DOI: 10.1039/C4RA12892C
HN
N
NH
NH2
O
N
N
OH
HN
N
N
HO
HO
HO
MeOH, TBAClO4
Stirring
Co(NO3)2, THF
N
N
HN
+
O-
Co3+ -O
TBANO , O
3
2
O
O
H
H
1
2
S
NH2
S
O
N
N
S
N
HO
N
N
HO
MeOH, TBAClO4
Stirring
S
Co(NO3)2, THF
TBANO , O
N
+
N
Co3+
O-
-O
3
2
3
4
NO2
S
S
N
N
NH2
O
N
N
S
N
N
HO
MeOH, TBAClO4
S
HO
Co(NO ) , THF
O-
3
2
Co3+ -O
+
N
Stirring
NO2
TBANO3, O2
NO2
O N
2
6
5
Scheme 1. Synthesis of receptors and cobalt complexes.
Among the various transition metals, cobalt was chosen because Co(III) was expected to act as a strong
2
3
Lewis acid and show good electrochemical responses to the binding of analytes. Powder X-ray diffraction
PXRD) was employed to analyze cobalt complexes 2, 4 and 6. Figure S1 illustrates that the diffraction patterns
(
of Co(III) complexes 2, 4 and 6 match neither receptors 1, 3 and 5 nor cobalt nitrate. The stoichiometry of
Co(III) complexes 2, 4 and 6 were confirmed by mass spectroscopy (Figures S8-S10). The analysis of mass
spectra of complexes 2, 4 and 6 showed a 2:1 ratio between the receptor and Co(III).
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Electrochemical detection of guanine
Benzimidazole/benzothiazole-based imine-linked Co(III) complexes 2, 4 and 6 were expected to have
high affinity towards anions and biomolecules by virtue of +3 oxidation state of cobalt and availability of H-
bonding sites (N or NH) of the complexes, as shown in Scheme 1. Platinum electrode was employed as working
electrode due to its robustness, versatility and behaviors as a noble metal. It provides more inert surface than
2
4
others. Linear sweep voltammetry (LSV) profile of complex 2 was recorded in the presence of various anions
-
-
-
-
-
-
-
-
(
F , Cl , Br , I , NO
3
, H
2
PO
4
, CH
3
COO and HSO
4
) and biomolecules (ATP, ADP, NAD, NADH, NADP,
O (8:2, v/v) solvent system (Figure S11). Addition of
adenine, guanine, cytosine and uracil) in a DMSO/H
2
guanine shifted the oxidation potential of complex 2. But the responses were not selective; other
anions/biomolecules also interfered. However, differential pulse voltammetry (DPV) profile of complex 2
showed a significant change upon addition of guanine (Figure 1A). It was observed that complex 2 has a peak
at -0.752 V. Addition of analytes, with the exception of guanine, did not cause any shift in the potential of
complex 2. The addition of guanine produced a significant change in the oxidation potential of complex 2. Two
new peaks arose at -0.552 V and -1.132 V with high current intensity. Furthermore, differential pulse
voltammograms of complex 2 and complex 2 + guanine were compared with guanine only (blank) as shown in
Figure 1B. The voltammogram of guanine alone did not show any resemblance to the other complexes, which
authenticated the binding between complex 2 and guanine.
Figure 1. (A) Changes in the DPV profile of complex 2 (10 µM) in the presence of various
tetrabutylammonium anion salts and biomolecules (30 µM) in a DMSO/H
Comparison of DPV profile of complex 2, complex 2 + guanine, and guanine only (blank) in a DMSO/H
8:2, v/v) solvent system.
2
O (8:2, v/v) solvent system; (B)
2
O
(
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Cyclic voltammetry (CV) profiles of complex 2 were recorded in the presence and absence of guanine
DOI: 10.1039/C4RA12892C
(
Figure 2). Complex 2 has an Epc and Epa at -0.766 V and -0.890 V; however, the addition of guanine led to
significant change in the anodic peak and a simultaneous shift in the cathodic potential, as shown in Figure 2.
The cyclic voltammograms of complex 2 at different scan rates (25, 50, 75, 100 and 120) were recorded. The
calibration plot was plotted between peak current and square root of scan rate and linear relationship was
observed (Figure 2B). The linear relationship indicates that the process is controlled by a diffusion process.
Figure 2. (A) Cyclic voltammogram of complex 2 (10 µM) with or without guanine (30 µM) in a DMSO/H
8:2, v/v) solvent system; (B) Calibration plot of complex 2 showing peak current versus square root of scan
rate (25, 50, 75, 100 and 120). Blue points represent current at -0.776 V and red points represent the current at -
.888 V.
2
O
(
0
To check the authenticity of binding, a titration was performed with complex 2 and guanine using a
DPV (Figure 3). The titration results revealed that the oxidation potential of complex 2 was shifted towards
higher potential, yielding a new peak at -0.552 V. Another peak was observed at -1.132 V. The plot was drawn
g
between peak current and concentration of guanine (C ) which showed a linear relationship in the concentration
range of 3.5-10 µM as shown in the inset of Figure 3.
The detection limit was calculated using the signal to noise ratio (3σ) method, and it was about 16.6 nM.
2
The linear regression equation was Ipa(µA) = -0.284C
g
(µM) + 0.802, R = 0.993. In addition, the recognition of
guanine in the presence of competing molecules was performed by measuring the current at -0.552 V (Figure 4).
For the selectivity study, a set of solutions were prepared that had an equal equivalent of guanine and other
competing molecules along with 10 µM complex 2. After incubation for 1 h, the DPV profile of these solutions
was recorded. A graph of the peak current at -0.552 V against various solutions was plotted. The graph showed
that complex 2 adequately recognized guanine even in the presence of other competing molecules, indicating
the high selectivity of complex 2.
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DOI: 10.1039/C4RA12892C
Figure 3. DPV curves of complex 2 (10 µM) in different concentrations of guanine (0 to 30 µM) in
DMSO/H O (8:2, v/v). The inset represents the linear relationship between the peak current at -0.552 V versus
2
the concentration of guanine.
Figure 4. Influence on the peak current of complex 2 (10 µM) in the presence of guanine along with equal
molar amounts of various competing anions and biomolecules in a DMSO/H O (8:2, v/v) solvent system.
2
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Binding interactions of benzothiazole-derived complexes 4 and 6 with a variety of anions and
DOI: 10.1039/C4RA12892C
biomolecules were accomplished using linear sweep voltammetry (LSV) and CV. All recognition studies of
2
complex 4 were recorded in a DMSO/H O (8:2, v/v) solvent system. Figure 5A exemplified the binding
behavior of complex 4 towards anions and biomolecules using LSV. Only guanine caused a change in the
oxidation potential of complex 4. From the titration experiments, it was observed that the addition of 5 µM of
guanine led to an enhancement at -0.435 V. Upon continuing the titration, the anodic peak at -0.435 V was
shifted toward lower potential and finally stabilized at -0.941 V. The addition of guanine was found to enhance
the current intensity only at -0.941 V (Figure 5B). A plot was drawn between the anodic peak current at -0.941
V versus concentration of guanine, and resulted in a linear relationship in the concentration range of 5 to 120
2
µM, as shown in inset of Figure 5B. The linear regression equation is Ipa(µA) = 0.225C
g
(µM) - 0.5994, R =
0
.984. The detection limit was approximately 13.4 nM, as calculated by the signal to noise ratio (3σ)
method.
Figure 5. (A) Linear sweep voltammogram of complex 4 (10 µM) in the presence of various
tetrabutylammonium anion salts and biomolecules (150 µM) in a DMSO/H O (8:2, v/v) solvent system; (B)
2
Successive changes in the LSV profile of complex 4 upon titration with guanine (0 to 150 µM); the inset
represents the linear relationship between the current intensity at -0.941 V and the concentration of guanine.
The CV also illustrated the high sensitivity of complex 4 towards guanine. A small amount of guanine
(
5 µM) triggered a huge change in the redox potential of complex 4 (Figure 6A). The cyclic voltammogram of
the blank solution (guanine only) was recorded and compared with complex 4 and complex 4 + guanine,
confirming the high affinity of complex 4 for guanine. The linear relationship was observed between peak
current and square root of scan rate, indicating a diffusion process (Figure 6B). To examine the interference of
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other analytes, recognition of guanine in the presence of an equal equivalent of other competing molecules was
DOI: 10.1039/C4RA12892C
performed (Figure 7). Complex 4 recognized the guanine even in the presence of other competing molecules.
Figure 6. (A) CV profile of complex 4 (10 µM), complex 4 + guanine (5 µM), and guanine only (50 µM)
blank) in a DMSO/H O (8:2, v/v) solvent system; (B) Calibration plot of complex 4 showing peak current
(
2
versus square root of scan rate (25, 50, 75, 100 and 120). Blue points represent current at -0.715 V and red
points represent the current at -0.851 V.
Figure 7. Effect on the peak current of complex 4 at -0.941 V in the presence of guanine along with equal
molar amounts of various competing anions and biomolecules in DMSO/H O (8:2, v/v).
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1
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2
Recognition studies of complex 6 were executed in a THF/H O (8:2, v/v) solvent system. Like complex
DOI: 10.1039/C4RA12892C
4
, LSV and CV were the principle analytical techniques used to explore its binding interaction with a variety of
anions and biomolecules. As shown in Figure 8A, the LSV profile of complex 6 was significantly altered in the
presence of guanine compared to other analytes. The guanine introduced a new peak at -0.252 V and shifted the
oxidation potential of complex 6 towards a higher potential. The peak currents for the interfering ions were
nearly the same as for guanine oxidation although the oxidation peak of guanine complex was shifted
significantly (Figure 8A). A new peak was observed at -0.252 V and the peak current increased linearly with
the addition of guanine (inset of Figure 8B). The linear range of detection, limit of detection (LOD) and
sensitivity of complex 6 towards guanine were calculated by generating a plot between peak current and
concentration of guanine at -0.252 V. A linear relationship was observed in the concentration range from 2.5 to
2
100 µM and the linear regression equation is Ipa(µA) = 0.142C
g
(µM) + 2.533, R = 0.994 as shown in the inset
of Figure 8B. The LOD was calculated using the signal to noise ratio (3σ) method and is about 11.3 nM. To
sense the guanine in a real environment, interference studies were performed. Analogous to other complexes, it
has good selectivity for guanine in a competing environment (Figure 9 and S12). An analysis of the interference
results revealed that complex 6 retains its affinity towards guanine even in the presence of other competing
molecules.
Figure 8. (A) Linear sweep voltammogram of complex 6 (10 µM) in the presence of various
2
tetrabutylammonium anion salts and biomolecules (150 µM) in a THF/H O (8:2, v/v) solvent system; (B)
Successive changes in the LSV profile of complex 6 upon titration with guanine (0 to 150 µM), the inset shows
the plot between the current at -0.252 V and the concentration of guanine.
1
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DOI: 10.1039/C4RA12892C
Figure 9. Interference study of complex 6 (10 µM) through monitoring the peak current at -0.252 V in the
presence of guanine along with equal molar of various competing anions and biomolecules in THF/H
v/v).
2
O (8:2,
For further clarification and to support the LSV results, binding studies were performed using the CV
technique. The cyclic voltammograms of complex 6, complex 6 + guanine, and guanine only were recorded in a
2
THF/H O (8:2, v/v) solvent system to prove the selectivity (Figure 10A). Complex 6 exhibited a well-defined
reversible redox couple with a cathodic potential at -1.023 V and anodic potential at -1.082 V. The addition of
guanine produced significant changes in redox potential at -0.813/-0.801 V with an enhancement in the anodic
peak current. Both oxidation and reduction peaks moved towards higher potential in the presence of guanine.
The voltammogram of a blank solution containing guanine only matched neither with complex 6 nor complex 6
+
guanine, supporting the specific binding between complex 6 and guanine. As an in-depth study, titration was
used and it was found that the anodic peak current was linearly dependent upon the concentration of guanine (5-
0 µM), as shown in the inset of Figure 10B. It is noteworthy to mention here that there is a large shift in the
8
anodic potential (ΔEpa = 275 mV) as well as in the cathodic potential (ΔEpa = 210 mV) during titration.
However, the cathodic peak current decreased and the anodic peak current increased with guanine addition. The
cyclic voltammograms of complex 6 were recorded at different scan rates (25, 50, 75, 100 and 120). The linear
relationship was observed between peak current and square root of scan rate, indicating a diffusion process
(Figure 11).
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Non-specific adsorption on working electrode may lead to electrode fouling and poor reproducibility.
DOI: 10.1039/C4RA12892C
To examine the antifouling property of the working electrode, cyclic voltammograms of complexes 2, 4 and 6
were recorded at different interval of time. The profiles remained the same even after 25 min with a small
decrease in current intensity, eliminating the possibility of non-specific adsorption on working electrode or
electron fouling (Figure S13). For optimization, pH titrations of complexes 2, 4 and 6 were performed. The
peak current of complexes 2, 4 and 6 remained constant and highest in the pH range of 4.5 to 10. Going beyond
these limits in either direction led to a decrease in peak current. Therefore, all studies were performed at pH 7.5
±
0.2.
Figure 10. (A) Comparison of the cyclic voltammogram of complex 6 (10 µM), complex 6 + guanine (80 µM),
and guanine only (50 µM) (blank) in a THF/H O (8:2, v/v) solvent system; (B) Gradual changes in the redox
2
potential of complex 6 (10 µM) upon successive addition of guanine (0 to 80 µM). The inset depicts the linear
relationship between the cathodic/anodic peak current at -0.813/-0.801 V and concentration of guanine (5 to 80
µM). Blue points represent the change in anodic current and red points represent the change in cathodic current.
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3

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DOI: 10.1039/C4RA12892C
Figure 11. Calibration plot of complex 6 showing peak current versus square root of scan rate (25, 50, 75, 100
and 120). Blue points represent current at -1.078 V and red points represent the current at -1.028 V.
Comparison of analytical performance of complexes 2, 4 and 6
Complexes 2, 4 and 6 are Co(III) complexes of benzimidazole/benzothiazole, which are enriched with
H-bonding sites. Consequently, the driving force behind the sensing of guanine is H-bonding. They are similar
in structure and all have similar selectivity towards guanine. It was interesting to compare analytical parameters,
such as LOD, the linear range of detection, and sensitivity of complexes 2, 4 and 6. A comparison was made
and it was found that all three have a limit of detection in the nanomolar range, as shown in Table 1. Among
them, complex 4 has a wide linear range of detection and good sensitivity. Furthermore, a comparison was
made between the present work and the reported literature, showing that the analytical data of complexes 2, 4
and 6 are comparable with previous publications. For reproducibility, the relative standard deviation (RSD) of
complex 2 (3.2 %), complex 4 (3.3 %) and complex 6 (3.1 %) was calculated (n = 7).
Table 1. Comparison of the analytical parameters of different methods for guanine detection.
Electrode
Technique*
DPV
Linear detection range LOD Ref.
nM)
(
Complex 2
Complex 4
Complex 6
3.5-10 µM
5.0-120 µM
2.5-100 µM
16.6
13.4
11.3
3
Present
work
Present
work
LSV
LSV
Present
work
1
1
9
6
Cobalt oxide nanostructure-modified Amperometry 40 nM-10 µM
glass carbon electrode
Cobalt
hexacyanoferrate-modified CV
0-4 µg
340
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DOI: 10.1039/C4RA12892C
carbon paste electrode
Cobalt(II) phthalocyanine-modified DPV
carbon paste electrode
Ionic liquid-carbon nanotube-gold DPV
nanoparticles composite
1
7
-
550
5
2
5
8 nM-2 mM
film coated electrode
2
2
6
7
Carbon screen-printed electrode
β-Cyclodextrin incorporated carbon DPV
nanotube-modified carbon paste
electrode modified electrode
LSV= Linear sweep voltammetry, DPV= Differential pulse voltammetry, CV= Cyclic voltammetry.
DPV
-
200
200
200 nM-20µM
*
Conclusions
Benzimidazole/benzothiazole-based imine-linked Co(III) complexes 2, 4 and 6 were prepared, and their
electrochemical properties were studied. Co(III) complexes 2, 4 and 6 showed high selectivity for the
electrochemical recognition of guanine. Complex 2 is a differential pulse voltammetric sensor while complexes
4
and 6 are linear sweep voltammetric sensors for nano-molar detection of guanine. The complexes recognize
guanine with high selectivity even in the presence of an equal equivalent of other competing molecules. The
complexes have a wide operational pH range. The novelty of this work is the unmodified electrode, which was
used for the detection of guanine with high selectivity, sensitivity and a low detection limit.
Supporting Information
PXRD, copies of NMR and mass spectra of receptors and their cobalt complexes.
Acknowledgments
This work was supported by the Korean and Indian Government for the India-Korea Joint Program of
Cooperation in Science & Technology (NRF-2011-0027710) and the CSIR project (project no:
01(2417)/10/EMR-II).
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