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Accepted Manuscript

Electrochemical sensing of purines guanine and adenine using single-walled carbon


nanohorns and nanocellulose

Túlio S. Ortolani, Tamires S. Pereira, Mônica H.M.T. Assumpção, Fernando C.


Vicentini, Geiser Gabriel de Oliveira, Bruno C. Janegitz

PII: S0013-4686(18)32824-X
DOI: https://doi.org/10.1016/j.electacta.2018.12.114
Reference: EA 33329

To appear in: Electrochimica Acta

Received Date: 11 September 2018


Revised Date: 21 December 2018
Accepted Date: 21 December 2018

Please cite this article as: Tú.S. Ortolani, T.S. Pereira, Mô.H.M.T. Assumpção, F.C. Vicentini, G. Gabriel
de Oliveira, B.C. Janegitz, Electrochemical sensing of purines guanine and adenine using single-
walled carbon nanohorns and nanocellulose, Electrochimica Acta (2019), doi: https://doi.org/10.1016/
j.electacta.2018.12.114.

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ACCEPTED MANUSCRIPT
Electrochemical sensing of purines guanine and adenine using single-
walled carbon nanohorns and nanocellulose

Túlio S. Ortolani1, Tamires S. Pereira1, Mônica H. M. T. Assumpção2,

Fernando C. Vicentini2, Geiser Gabriel de Oliveira3 and Bruno C. Janegitz1,*

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Department of Nature Sciences, Mathematics and Education, Federal University of São

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Carlos, 13600-970 Araras, SP, Brazil.

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Center of Nature Sciences, Federal University of São Carlos, Rod. Lauri Simões de
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Barros km 12, Buri, SP, Brazil.
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Universidade Federal do Tocantins, Campus Gurupi, 77402-970, Gurupi, TO, Brazil
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Corresponding author: Tel +55 19 3543 7601

E-mail address: brunocj@ufscar.br (Bruno Campos Janegitz)


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Abstract

In this study, we report an electrochemical study based on nanocellulose (NC)

and single-walled carbon nanohorns (SWCNH). SWCNH and NC ensure large surface

area, good conductivity, high porosity and chemical stability, becoming attractive for

electrodes. The materials were characterized by X-ray diffraction (XRD), Fourier

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transform infrared (FTIR), Scanning Electron Micrograph (SEM), Transmission

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electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential. Using

XRD and FTIR it was possible to observe particular characteristics of NC and SWCNH.

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The presence of dahlia-like assemblies on the NC surface was observed by MEV and

TEM. Then, we investigated the electrochemical behavior of NC-SWCNH, which

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showed the excellent results when it was used guanine and adenine, as proof of concept,
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by using cyclic and linear sweep voltammetry (LSV). LSV was also employed for

simultaneous detection resulting in limits of detection of 1.7 × 10−7 mol L−1 and
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1.4 × 10−6 mol L−1, for guanine and adenine, respectively. In addition, the proposed
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electrode was applied for determination of both bases in synthetic human serum and fish
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sperm. We demonstrate that it is possible to use NC, a renewable material, in

conducting thin films with SWCNH, and due to simplicity in the preparation and high
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conductivity, this new thin film could be extended for others electrochemical purposes

such as sensing and biosensing.


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Keywords: electrochemical detection, single-walled carbon nanohorns, nanocellulose;

guanine and adenine determination.

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1. Introduction

Among the various modifiers, carbon nanotubes [1-3], graphene [4, 5], metallic

nanoparticles [6-10] and artificial polymers [11, 12] have been utilized to decorate

electrode. However, recently, news structures such as single-walled carbon nanohorns

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(SWCNH) and nanocellulose (NC) have attracted much attention due to their high

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surface area [13-15].

SWCNH can be described as a kind of carbon material with horn-shaped

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graphitic tubes having diameters of 2-5 nm and lengths of 40-50 nm. They are derived

from single walled carbon nanotubes and terminated by a five-pentagon conical cap

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with a cone opening angle of ~19º and about 2000 of them assemble radially to form a
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spherical aggregate with diameters of about 100 nm [13, 16, 17]. SWCNH are cost-
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effectively produced by direct vaporization of pure graphite without the use of metal

catalysts what result in the absence of metal contamination and consequently no need of
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purification [14, 18, 19]. This kind of material ensures extremely large surface area,
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high dispersibility, peculiar structure and morphology, excellent conductivity,

hydrophobic nature of its surface, chemical stability and fast mass transport. As a result,
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SWCNH have been used in many applications such as electrodes [13, 14, 19, 20].

Cellulose is a polysaccharide considered the major biodegradable polymer found


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in structural components of the cell wall of green plants. Nanocellulose are cellulose

elements with at least one dimension smaller than 100 nm obtained from renewable

polymers via acid hydrolysis. Hence, NC is a natural fiber which can be extracted from

plant cell wall and it has been successfully isolated from cellulose obtained from

sugarcane [15, 21-25]. NC exhibits high strength, high stiffness, high surface area, good

mechanical and rheological properties and hydroxyl groups. Additionally with low

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density, lightweight, high porosity, lack of toxicity and biodegradability NC based

materials become high attractive for many applications such as biomedical [15, 21, 22,

25].

Deoxyribonucleic acid (DNA) bases detection is of great significance for clinical

diagnosis, insight into fundamental mechanism of genetic information and physiological

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and pharmacological studies [11, 26]. DNA encodes genetic information via unique

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combinations of four DNA bases: guanine, adenine, thymine and cytosine. Thus,

analyzing individual base composition the DNA can be measured since the mole

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percentage of guanine is equal to cytosine and that of adenine is equal to thymine [27].

Therefore, adenine and guanine are two important constituent parts of both

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deoxyribonucleic acid and ribonucleic acid (RNA) and play key roles in the storage of
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genetic information and also in protein biosynthesis [28, 29]. Thus, selective and
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sensitive detection of these bases provides valuable insights in fundamental fields such

as understanding of DNA sequence, oxidative damage and hybridization and protein


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metabolism in cells [28]. Moreover, abnormal changes of DNA bases suggest the
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deficiency of the immunity system what could indicate the presence of some diseases

such as cancer, Alzheimer, epilepsy and HIV infection [1, 11, 30].
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Some analytical methods have been developed to the determination of guanine

and adenine. Among them are mass spectrometry [31], chemiluminescence [32], HPLC
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[33] and electrophoresis [34]. However, most of these methods involve time-consuming

sample pretreatments, complicated operations and/or expensive instruments. Thus,

electrochemical methods are in forefront considering its low costs, rapidity,

convenience, excellent sensitivity and selectivity [1, 30]. In fact electrochemical method

has been widely used in the determination of guanine and adenine [6, 27, 29, 30, 35-37]

however, it is difficult to obtain accurate oxidation signals of these bases because of

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their extremely positive oxidation potentials, weak direct electron transfer capacity and

irreversible adsorption on traditional electrode surfaces, which leads to low sensitivity

and reproducibility. In order to overcome these drawbacks, many materials have been

employed to modify electrodes and so obtain a wide potential window, high

electrocatalytic activity and antifouling property [1, 11, 38].

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Aiming of thin film for electrochemical purposes, we developed a new

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composite by using on NC and SWCNH, which was applied toward guanine and

adenine determinations. It is expected that this material could provide high catalytic

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activity and antifouling properties that are of great interest in electroanalysis.

2. Experimental
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2.1 Reagents and solutions
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All chemical were of analytical grade and used without any further treatment or

purification. SWCNH, adenine and guanine were purchased from Aldrich as well as
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adenine and guanine bases. Phosphate buffer, KCl and H2SO4 were purchased from
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Synth. For adenine and guanine solutions preparation, the bases were first dissolved in

ultra-pure water at 80 ºC and maintained in a hot bath for 40 minutes. In all prepared
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solution ultra-pure water was used as solvent. Sugarcane bagasse was purchased at

Porto Ferreira city (São Paulo state) in a local market.


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2.2 Nanocellulose preparation

The NC preparation was done basing on Teixeira et al [39] and Lee et al [40].

Firstly, 5 g of sugarcane bagasse were washed and dried at 55 ºC until constant weight.

Then, the bagasse was powdered and put in a flask containing 100 mL of NaOH (5

wt%) and 100 mL of hydrogen peroxide (11% (v/v)) solutions, in order to remove lignin

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and hemicelluloses. This process was taken at 40 ºC and magnetic stirring for 90

minutes. After that, the final solution was filtered and dried at 55 ºC until constant

weight. Subsequently, 5 g of the bagasse previously treated was dispersed in 100 mL of

6 mol L−1 sulfuric acid at 45 ºC and vigorously stirred for 30 min. Next, 500 mL of cold

deionized water (4 oC) were added in order to stop the reaction. Then, the final solution

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was centrifuged and the non-reactive sulfate groups removed. The solids obtained were

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washed with water until pH reached to 7 and the neutral suspension was then

ultrasonicated in order to obtain a homogenized solution.

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2.3 Electrode characterization

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For electrochemical characterization of the prepared electrode it was used
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0.1 mol L−1 KCl as supporting electrolyte in the presence of equimolar mixtures of
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1.0 × 10−3 mol L−1 K3[Fe(CN)6] and 1.0 × 10−3 mol L−1 K4[Fe(CN)6]. For the purines

determinations 0.2 mol L−1 phosphate buffer solution was used as supporting
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electrolyte.
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2.4 Analytical procedure

Electrochemical measurements were conducted at room temperature using a


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potentiostat/galvanostat PGSTAT 204 Metrohm (ECO CHEMIE) and NOVA 2.1


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software. A conventional three-electrode electrochemical cell composed of a platinum

electrode as counter electrode, a glassy carbon electrode (GCE, HTW/nfinitu Tech

LLC) of 3 mm as work electrode and an Ag/AgCl (3.0 mol L−1 KCl) as reference

electrode. The work electrodes were prepared by dispersing 1.0 mg of SWCNH in 1.0

mL of the nanocellulose solution. The resultant solution was put in an ultrasonic bath

for 20 min. Thus, 3 µL of this ink was pipetted on the work electrode and dried at room

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temperature during 2 h. After dry more 3 µL was added and dried for more 2 h. The

modified electrode was designed SWCNH –NC/GCE.

2.5 Materials characterization

The prepared materials were characterized by Fourier transform infrared (FTIR)

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using a Tensor II (Bruker) spectrophotometer in the range of 500 a 4000 cm−1. For

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dynamic light scattering (DLS) and zeta potential analyses it was employed a Zetasizer

ZS 90 (Marvern instruments). Scanning electron micrographs (MEV) were performed

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using Scanning Electron Microscope XL-30 FEG Philips® while transmission electron

microscopy (TEM) images were obtained using a Jeol JEM 1200 EX-II. XRD analysis

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were also obtained and recorded in the range of 2θ = 20° to 90° with a step size of 0.02°
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and a scan time of 10 deg/min per step using a Rigaku diffractometer model Miniflex II
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with a Cu Kα radiation source (0.15406 nm).


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2.6 Samples
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The sperm fish was collected in CEPTA/ICMBio in Pirassununga - São Paulo. This

sample was collected from fish Astyanax Bimaculatus, popularly known as Lambari,
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populary found in different areas in Brazil. The 1.0 mL of sample was diluted in 1.0 mL

of 1.0 mol L−1 sulfuric acid and heated to 90°C by 90 min in closed bottle. It was cooled
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at room temperature and then it was added 1.0 mL of 1.0 mol L−1 sodium hydroxide for
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neutralization. Finally, 5.0 mL of 0.2 mol L−1 phosphate buffer (pH 8.6) to perform the

measures. The synthetic serum was also utilized for guanine and adenine detection [41].

In the volume of 1.0 mL of this sample was added amounts of guanine and adenine,

which were diluted in electrochemical cell for the electrochemical measurements. When

it was not in use, the prepared samples were kept in refrigerator at 4°C.

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3. Results and discussion

3.1 DLS, FTIR, SEM, TEM and XRD analysis

DLS is a widely used method to obtain nanoparticles distribution in suspensions.

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Fig. 1(a) and (b) shows the DLS analysis. Using this technique, it was possible to

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observe that NC nanoparticles are in the range of 32 nm – 160 nm with a non-normal

distribution. However, the composite material of SWCNH and NC (NC-SWCNH)

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showed the nanoparticles in the range of 105 nm – 526 nm and a normal distribution.

The zeta potential has been reported as a good indicator of the acid hydrolysis

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effectiveness and provides information about stable and colloidal suspension.
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According to Benini et al [22] generally, values lower than – 15 mV represent the
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beginning of particle size agglomeration while values higher than – 30 mV mean that

there is sufficient mutual repulsion resulting in colloidal stability. According to Fig.


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1(c), the obtained value for NC-SWCNH is almost – 30 mV, resulting in a good
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stability. Besides, NC shows a zeta potential of – 19 mV resulting in a more

agglomerated material. Thus, knowing that the higher zeta potential in suspension the
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higher stability of the suspension and consequent formation of a colloidal suspension

[22, 42] it is possible to affirm that the addiction of SWCNH increases the NC stability.
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It is worth noting that SWCNH DLS analysis was not performed since its dispersion

ability in aqueous solution is rather pour due to the strong π–π interaction [42].

Fig. 2 shows FTIR spectra of NC, SWCNH and NC-SWCNH. A broad band in

the region of 3350 cm−1 is observed for all materials. This indicates the stretching

vibration of O-H groups involved in intra and intermolecular hydrogen bonds present in

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cellulose and also absorbed/adsorbed water molecules [22, 43]. The bands observed

around 2900 cm−1 are associated to C-H stretching vibration in cellulose and

hemicellulose and peaks located at 1427 cm−1 could be related to the C=C stretching

and/or CH2 symmetric bending of methylene groups of cellulose due to crystallinity

band [15, 21, 22]. It is important to highlight that these bands do not appear on the

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SWCNH spectrum. Besides, they appear on the NC-SWCNH, indicating the presence

NC on the SWCNH. Peaks observed at 1030 cm−1 and 896 cm−1 could be associated to

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C-O stretching and B-glycosidic links between the glucose units of cellulose,

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respectively [21, 22]. Considering SWCNH spectrum, the band observed at 1637 cm−1

can be ascribed to O-H bending of adsorbed water [43].

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The morphology of different electrodes was characterized by using SEM and
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TEM, which the results are shown on Fig. 3. According to Ferreira et al [44] raw
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bagasse has relatedly smooth and homogeneous external cell tissues on its surface.

However after treatment some cells can be destroyed leaving to rougher surface what
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can be related to the partial removal of lignin and hemicellulosed during the NC
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preparation (Fig. 3(a) and 3(d)). According to Chen et al [45], SWCNH formed dahlia-

like assemblies with a diameter about 70 nm. Considering MEV magnitude, it is


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possible to observe globular and highly compact aggregate structure (Fig. 3(b))

nevertheless, considering TEM image (Fig. 3e) dahlia structure is clearly observed.
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Considering the NC-SWCNH SEM and TEM image (Fig. 3(c) and 3(f)) it is possible to

observe that the NC surface is covered by SWCNH, with the formation of some

agglomerates, but without apparent change in morphology. A similar behavior was

observed by Youssef et al [46] who evaluated the functionalization of cellulose bagasse

and rice straw with PANi at different concentrations.

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X-ray diffraction data are disposed on Fig. 4. NC showed two intense peaks at

16º and 22º which represent the typical cellulose structure [21, 22, 47]. The first one at

16º corresponds to crystallographic planes, overlapping (110) and (110) peaks, [21, 22]

while the second one at 22º corresponds to the (200) plane, [21, 22]. The SWCNH

showed a characteristic peak at 26º corresponding to the (022) reflection of graphite and

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another one at around 43º which can be attributed to the (002) reflection of graphite as

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already observed before [45, 48]. It is important to highlight that the NC-SWCNH

showed the characteristic peak of SWCNH at 26º and the one at 16º and 22º observed

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for NC.

3.2 Electrochemical characterization


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Fig. 5 shows the voltammetric profiles of two types of work electrodes: (a) GCE

and (b) NC-SWCNH/GCE in a 0.1 mol L−1 KCl and 1.0 × 10−3 mol L−1 equimolar
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mixture of K3[Fe(CN)6]/K4[Fe(CN)6] and different scan rates ranging from 50 to 300


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mV s−1. In this Figure it is possible to observe a linear increase of both anodic and
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cathodic peak current with the increase of the scan rate for both electrodes. By the

Randles-Sevcik equation, it is possible to estimate the electroactive areas. According to


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this estimation, 0.04 cm2 was achieved for GCE and 0.13 cm2 for NC-SWCNH/GCE, a
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value higher than obtained for GCE, what can be explained by the high surface area of
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NC and SWCNH [13-15]. Additionally, it can be seen that NC-SWCNH showed higher

oxidation and reduction peaks (Fig 5c), but GCE presented a lower peak-to-peak

separation (∆E=76 mV) as compared within the modified electrode (∆E=102 mV).

However, for purines basis the proposed electrode presented electrocatalytic effect what

is really desired for the purines determination as proposed by Wang et al [1] and

Prathap et al [11] as observed next.

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Electrochemical studies were performed in presence of guanine and adenine

(individually) by using different electrodes. Firstly, Fig. 6(a) displays cyclic

voltammetric profiles obtained for GCE, NC/GCE and NC-SWCNH/GCE in

9.9 × 10−5 mol L−1 guanine solution and 0.2 mol L−1 phosphate buffer at a scan rate of

50 mVs−1. Considering this Figure, NC-SWCNH showed the highest peak currents at

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0.74 V. This result indicated the electrocatalytic activity of NC-SWCNH toward

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guanine oxidation either. This effect can be associated to a synergic effect of NC and

SWCNH. The same experiments were taken considering adenine.

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Fig. 6(b) displays cyclic voltammetry profiles obtained for GCE, NC/GCE and

NC-SWCNH/GCE in 2.0 × 10−4 mol L−1 adenine solution and 0.2 mol L−1 buffer

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phosphate at a scan rate of 50 mVs−1. Once more, NC-SWCNH/GCE showed the
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highest peaks current at 1.01 V. This result indicated the electrocatalytic activity of NC-
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SWCNH toward adenine oxidation what can be explained by a synergic effect between

NC and SWCNH.
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None process was observed in cathodic direction which means that the charge
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transfer process is irreversible for both analytes. Hence, the proposed mechanism for the

oxidation of guanine and adenine according to Abbaspour and Noori [49] is represented
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in Scheme 1.
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Scheme 1. Schematic representation of the oxidation of guanine and adenine.

Taking into account the highest electrocatalytic activity of NC-SWCNH toward

guanine and adenine this material was employed toward simultaneous determination of

guanine and adenine. Fig. 6(c) shows cyclic voltammetric profiles obtained for GCE,

NC/GDE and NC-SWCNH/GDE in 9.9 × 10−5 mol L−1 guanine solution,

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2.0 × 10−4 mol L−1 adenine solution and 0.2 mol L−1 buffer phosphate at a scan rate of

50 mVs−1. Once again, the NC-SWCNH show better results when compared to pure

GCE and NC/GCE. Thus, NC-SWCNH show a peak of 6 µA at 0.74 V associated to the

guanine oxidation and another peak of 34 µA at 1.04 V associated to adenine oxidation.

It is also possible to affirm that the oxidation of guanine and adenine is irreversible

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since there is no peak in the cathodic region.

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We also investigated the simultaneous detection of guanine and adenine using

Linear Sweep Voltammetry (LSV). This experiment was conducted using GCE and

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NC-SWCNH/GCE in 0.2 mol L−1 phosphate buffer (pH 7.0) solution as supporting

electrolyte in presence of 9.9 × 10−5 mol L−1 guanine solution and 2.0 × 10−4 mol L−1

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adenine solution. As can be seen in Fig. 7, both analytes presented a well-defined
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oxidation process for NC-SWCNH/GCE with anodic peak potentials at 632 mV
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(guanine) and 969 mV (adenine). For GCE the anodic peak potential were at 698 mV

and 998 mV for guanine and adenine, respectively. In addition, the NC-SWCNH/GCE
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presented an anodic peak current of 3.0 µA (guanine) and 60.4 µA (adenine) whereas
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the GCE the Ipa was 0.3 µA and 6.2 µA for guanine and adenine, respectively, an

increase in the anodic current of 10 (guanine) and 9.7-fold (adenine) higher than the
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unmodified sensor. This result demonstrates that the proposed sensor leads to an
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electrocatalytic effect, since there was a decrease in the anodic potential and an increase
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of the analytical signal in the simultaneous oxidation of guanine and adenine.

The dependence of the anodic peak current for simultaneous oxidation of

guanine and adenine by LSV scan rate was evaluated in the range from 10 to

300 mV s−1 in the presence of 9.9 × 10−5 mol L−1 guanine solution and

2.0 × 10−4 mol L−1 adenine solution in a 0.2 mol L−1 phosphate buffer solution (pH 7.0).

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An increase in the analytical signal with increase of scan rate (data not shown) was

observed up to 200 mV s−1. Thus, this scan rate was selected for next studies.

The effect of the pH on the anodic peak current of the NC-SWCNH/GCE was

evaluated in presence of 9.9 × 10−5 mol L−1 guanine solution and 2.0 × 10−4 mol L−1

adenine solution in a 0.2 mol L−1 phosphate buffer solution in a pH range from 4.3 to

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8.6. An increase of pH of the solution leads to a shift of the peak potentials to more

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negative potentials, in addition, the higher analytical signal was also obtained in basic

medium (pH 8.6). Then phosphate buffer solution (pH 8.6) was selected for further

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studies.

Then, the effect of accumulation time was investigated in the range from 30 to

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180 s. The anodic peak current increased up to 90 s for guanine and up to 60 s for
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adenine (data not shown). Since the detection of guanine using the NC-SWCNH/GCE
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generates a smaller analytical signal, the accumulation time of 90 s was selected for the

simultaneous determination.
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LSV was employed for the individual determination of guanine and adenine
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using a NC-SWCNH/GCE, taking into account the linear range, sensitivity, and limit of

detection. The analytical curves were carried out by adding different concentrations of
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the standard solutions to the 0.2 mol L–1 phosphate buffer (pH 8.6) solution.

Under optimized parameters, analytical curves were constructed for guanine


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and adenine, individually. The analytical curve for guanine was linear from 7.4 × 10−7
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to 6.4 × 10−6 mol L–1 with the following equation: Ip (µA) = −2.91 × 10−6 + 0.061

[guanine] (µmol L–1) (r = 0.998), with limit of detection of 1.5 × 10−7 mol L–1, as

calculated as 3 × SD/m, where SD is the standard deviation of analytical curve and m is

the slope of the analytical curve. The obtained linear range for adenine was from 7.4 ×

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10−6 to 2.1 × 10−4 mol L–1 with the following equation: Ip (µA) = 1.39 × 10−4 + 2.65 ×

10−5 log[adenine] (µmol L–1) (r = 0.994), with limit of detection of 1.9 × 10−7 mol L–1.

Then, the proposed NC-SWCNH/GCE using LSV was applied for the

simultaneous determination of guanine and adenine. Fig. 8 presented the LS

voltammograms obtained and the respective analytical curves (inset). For guanine, the

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analytical signal increases from 7.4 × 10−7 to 6.4 × 10−6 mol L–1 and the obtained

equation was Ip (µA) = − 6.27 × 10−7 + 1.68 [guanine] (µmol L–1) (r = 0.999). The

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anodic current increases from 7.4 × 10−6 to 2.1 × 10−4 mol L–1 for adenine, the equation

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obtained was Ip (µA) = 3.11 × 10−4 + 6.33 × 10−5 log[adenine] (µmol L–1) (r =

0.937).The limits of detection were 1.7 × 10−7 and 1.4 × 10−6 mol L−1, for guanine and

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adenine, respectively. The proposed electrode was applied for the determination of the
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purine bases in synthetic serum and fish sperm, which it was obtained recuperation
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ranging from 84 to 112% as shown in the Table 1.


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Table 1: Determination of guanine and adenine by using NC-SWCNH/GCE

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%

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Guanine Adenine Guanine proposed Adenine proposed % recovery
Sample recovery
[added] (µmol L−1) [added] (µmol L−1) electrode (µmol L−1) electrode (µmol L−1) Adenine
Guanine

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Synthetic 4.00 12.5 3.70 12.2 92.5 97.6

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Serum 4.40 11.3 3.69 12.2 83.9 108

Fish 3.80 10.8 3.70 12.0 97.4 111

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sperm

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As presented in Table 2, the proposed NC-SWCNH/GCE sensor presented a

similar limit of detection than proposed by Wang et al.[6], Fan et al. [35] and Ensafi et

al. [37], a lower limit of detection than sensors proposed by Yari et al. [50, 51] and

higher than proposed by other works [28-30, 36, 38, 52] used in the simultaneous

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detection of purines. Furthermore, the sensitivity of the proposed method for guanine

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determination is higher than those of [6, 28-30, 35, 37, 38, 50, 51] and lower than [36,

52], however, the electrodes developed by Wu et al. [36] and Shahrokhian et al.[52] had

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a shorter linear range. It was not possible to compare the sensitivity of the proposed

method for the adenine determination because the analytical curve was linearized by the

logarithm of the concentration.


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It is interesting to highlight that we used NC, a renewable material that can be
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applied for other carbon materials immobilization. In addition, the proposed electrode it

is easy to prepare/use and has high stability attributed to the compatibility of NC-
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SWCNH.
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Table 2: Comparisons of various electroanalytical methods proposed for simultaneous detection of guanine and adenine

Linear range (µmol L−1) Limit of detection (µmol L−1) Sensitivity (A mol−1 L)

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Sensor Reference
guanine adenine guanine adenine guanine adenine

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Cu@Ni-MWCNTs/GCE(a) 1.0–180 2.0–150 0.17 0.33 0.419 0.438 Wang et al., 2018 [6]

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Chit-CNF/GCE(b) 0.2–50 0.2–50 0.046 0.074 0.014 0.005 Thangaraj et al., 2014 [28]

PPyox-GR/GCE(c)

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0.04–100 0.06–100 0.01 0.02 0.137 0.119 Gao et al., 2014 [29]

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MoxC@C/GCE(d) 0.03–122 0.02–122 0.0085 0.008 0.11 0.10 Zhang et al., 2018 [30]

TiO2-G/GCE(f) 0.5–200 0.5–200 0.15 0.10 0.115 0.108 Fan et al., 2011[35]

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GS/GCE(g) 0.025–2.5 0.05–2.5 0.01 0.01 5.9 6.6 Wu et al., 2015 [36]

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[PW11NiO39]5−/PDDA@CNTs/GCE(h) 0.4–76 0.4–136 0.1 0.24 0.696 0.581 Ensafi et al., 2015 [37]

BDDE(e) 0.3–19
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0.3–19 0.037 0.019 0.019 0.045 Švorc et al., 2014[38]
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Fe3O4NPs/MWCNT(i) 0.05–8 0.01–10 0.001 0.005 3.31 3.17 Shahrokhian et al., 2012[52]
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TAN-AgNP-PANT/CPE(j) 0.9–140 1.0–200 3.00 2.8 0.021 0.044 Yari et al., 2016 [50]
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MWNCNT-Fe3O4@PDA-Ag/CPE(k) 8–130 10–120 1.47 5.66 0.300 0.438 Yari et al., 2016[51]

SWCNH-NC/GCE 0.74–6.4 7.4–210 0.17 1.4 1.68 - This work

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(a)
copper-nickel nanoparticles-multiwalled carbon nanotubes modified GCE.
(b)
chitosan-carbon nanofiber modified GCE.
(c)

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overoxidized polypyrrole-graphene modified GCE.
(d)
molybdenum carbide nanosphere modified GCE.
(e)
BDDE boron-doped diamond electrode.

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(f)
TiO2 nanoparticles-graphene nanocomposite modified GCE.

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(g)
graphene nanosheets modified GCE.
(h)
polyoxometalate modified poly diallyl dimethyl ammonium chloride-multiwalled carbon nanotubes modified GCE.
(i)
Fe3O4 nanoparticles-multiwalled carbon nanotubes modified GCE.

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(j)
1,3,5-Trithiane Ag nanoparticles- polyaniline nanofibers modified carbon paste electrode.

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(k)
Multiwalled carbon nanotubes-Ag nanoparticles modified carbon paste electrode.

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The stability studies were performed by using one thin film for 5 consecutive

days (one measurement per day) in the presence of of 1.8 × 10−6 mol L–1 guanine

solution and 1.8 × 10−5 mol L–1 adenine solution in a 0.2 mol L–1 phosphate buffer (pH

8.6) solution. For guanine the response decreased 27% and for adenine the response

decreased 12% after this period (see Fig. S1, in the Supplementary Material). However,

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it is important to highlight the proposed thin film is easy to prepare and could be

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replaced before each measurement. The repeatability studies of the NC-SWCNH/GCE

was investigated using concentrations of 9.9 × 10−5 mol L–1 guanine solution and 2.0 ×

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10−4 mol L–1 adenine solution in a 0.2 mol L–1 phosphate buffer (pH 8.6) solution.

Therefore, we performed intra-day (n = 3) and inter-day (n = 4) repeatability studies.

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The RSD values obtained for intra-day studies were 2.2% for guanine and 3.5% for
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adenine; in addition, for inter-day experiments the obtained RSD values were 2.6% and
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4.7% for guanine and adenine, respectively. A high repeatability of the proposed

electrode was obtained, due the fact of simplicity of preparation.


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4. Conclusions

In this work, we report the use of NC-SWCNH/GCE toward guanine and


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adenine determinations. The prepared electrode showed highly sensitive and high

electrocatalytic activity toward simultaneous determination of guanine and adenine. The


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proposed NC-SWCNH/GCE sensor presented lower limits of detection when compared


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with other electrodes used in the simultaneous detection of purines. In addition, the

proposed electrode is easy to prepare, presented great repeatability and utilize NC, a

renewable material.

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Acknowledgements

The authors gratefully acknowledge the financial support granted by CAPES,

FAPESP (2017/21898-6) and CNPq (408430/2016-8 and 407674/2016-0).

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Legend of figures

Fig. 1: Size distribution obtained by DLS for (a) NC, (b) NC-SWCNH and (c) Zeta

potential analyses for NC and NC-SWCNH.

Fig. 2: FTIR spectra of NC, SWCNH and NC-SWCNH.

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Fig. 3: SEM images with 1 µm of scale for (a) NC, (b) SWCNH and (c) NC-SWCNH

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and TEM images with different magnitudes for (d) NC, (e), SWCNH and (f) NC-

SWCNH.

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Fig. 4: XRD patterns for NC, SWCNH and NC-SWCNH.

Fig. 5: Cyclic voltammetry profiles obtained for (a) GCE and (b) NC-SWCNH/GCE in

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0.1 mol L−1 KCl and 1.0 × 10−3 mol L−1 [Fe(CN)6]3−/4− equimolar and different scan
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rates. (c) Comparison between GCE (blue) and NC-SWCNH/GCE (orange) in 0.1 mol

L−1 KCl and 1.0 × 10−3 mol L−1 [Fe(CN)6]3−/4− equimolar at scan rate of 50 mV s-1.
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Fig. 6. Cyclic voltammograms obtained for blank (NC-SWCNH/GCE, green curve),


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GCE (blue curve), NC/GCE (black curve) and NC-SWCNH/GCE (red curve) in 0.2 mol
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L−1 phosphate buffer (pH 7.0) solution as support electrolyte in presence of (a) 9.9 ×

10−5 mol L−1 guanine solution, (b) 2.0 × 10−4 mol L−1 adenine solution and (c) 9.9 ×
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10−5 mol L−1 guanine solution and 2.0 × 10−4 mol L−1 adenine solution. v = 50 mVs−1.

Fig. 7. Linear sweep voltammograms obtained for GCE (black curve) and NC-
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SWCNH/GCE (red curve) in 0.2 mol L−1 phosphate buffer (pH 7.0) solution as support
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electrolyte in presence of 9.9 × 10−5 mol L−1 guanine solution and 2.0 × 10−4 mol L−1

adenine solution. v = 50 mVs−1.

Fig. 8. LS voltammograms obtained using the NC-SWCNH/GCE for various

concentrations of guanine (a-e: 7.64 × 10−7; 1.22 × 10−6; 2.10 × 10−6; 3.46 × 10−6; 6.35 ×

10−6 mol L–1) and adenine (f-j: 7.40 × 10−6; 1.84 × 10−5; 3.68 × 10−5; 7.32 × 10−5; 2.10 ×

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10−4 mol L–1) and supporting electrolyte: 0.2 mol L–1 phosphate buffer (pH 8.6). v = 200

mV s−1. Accumulation time = 90s. Inset: analytical curves obtained for (i) guanine and

(b) adenine.

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