Electrophoresis 2017, 38, 2733–2740 2733

Eiva Natiele Tiago da Silva1 Research Article

Jacqueline Marques Petroni1
Bruno Gabriel Lucca2
Valdir Souza Ferreira1
1 Instituto de Quı́mica,
Universidade Federal de Mato
Grosso do Sul, Campo Grande,
Brazil
2 Departamento de Ciências
Naturais, Universidade Federal
do Espı́rito Santo, São Mateus,
Brazil
Received April 10, 2017
Revised July 31, 2017
Accepted August 4, 2017
Pencil graphite leads as simple
amperometric sensors for microchip
electrophoresis
In this work we demonstrate, for the first time, the use of inexpensive commercial pencil
graphite leads as simple amperometric sensors for microchip electrophoresis. A PDMS
support containing one channel was fabricated through soft lithography and sanded pen-
cil graphite leads were inserted into this channel to be used as working electrodes. The
electrochemical and morphological characterization of the sensor was carried out. The
graphite electrode was coupled to PDMS microchips in end-channel configuration and
electrophoretic experiments were performed using nitrite and ascorbate as probe analytes.
The analytes were successfully separated and detected in well-defined peaks with satisfac-
tory resolution using the microfluidic platform proposed. The repeatability of the pencil
graphite electrode was satisfactory (RSD values of 1.6% for nitrite and 12.3% for ascorbate,
regarding the peak currents) and its lifetime was estimated to be ca. 700 electrophoretic
runs over a cost of ca. $ 0.05 per electrode. The limits of detection achieved with this
system were 2.8 ␮M for nitrite and 5.7 ␮M for ascorbate. For proof of principle, the pencil
graphite electrode was employed for the real analysis of well water samples and nitrite
was successfully quantified at levels below its maximum contaminant level established in
Brazil and US.

Keywords:
Carbon electrode / Electrochemical detection / Environmental analysis /
Microfluidics / PDMS devices DOI 10.1002/elps.201700160

 Additional supporting information may be found in the online version of this

article at the publisher’s web-site

1 Introduction employed for the fabrication of ME devices, the most used

The development of miniaturized analytical systems has
been one of the most explored scientific advances in the
last years [1, 2]. In this context, microchip electrophoresis
(ME) devices are a very interesting option to perform inno-
vative analytical procedures and offer features such as faster
analysis, low waste generation and low consumption of sam-
ples and reagents [3]. The use of ME devices have been ex-
plored in various applications such as clinical analysis [4],
monitoring of environmental pollutants [5], food analysis [6],
biological applications [7] and detection of biohazards [8]
and explosives [9]. Among the substrate materials usually
Correspondence: Professor Dr. Bruno Gabriel Lucca,
Departamento de Ciências Naturais, Universidade Federal
do Espı́rito Santo, São Mateus, ES, 29932-540, Brazil
E-mail: bruno.lucca@ufes.br
Abbreviations: AD, amperometric detection; MCL, maximum
contaminant level; PGE, pencil graphite electrode; PGWE,
pencil graphite working electrode
are glass and polymers such as polydimethylsiloxane (PDMS)
and polymethylmethacrylate (PMMA) [10]. PDMS has as ad-
vantages its low cost, the easiness for fabrication of the de-
vices and the possibility to easily integrate electrodes into the
microfluidic systems [11].
Electrochemical techniques, such as amperometry, have
been frequently coupled to ME and employed for the detec-
tion of several electroactive substances without the use of
derivatization steps. Besides offer satisfactory sensitivity and
selectivity, the amperometric detection (AD) also enables the
miniaturization of the instrumentation and allows that the
detection electrodes be fabricated using the same microfabri-
cation methods employed for production of the microfluidic
devices [12–14]. The use of carbon-based electrodes as am-
perometric sensors in ME has been widely reported [15–19].
Carbon electrodes are interesting for detection in ME-AD due
to features such as the large potential window, favored kinetic
for the oxidation of organic substances and reduced fouling
when applied for detection of biological analytes [20].
Colour Online: See the article online to view Figs. 1, 3–5 in colour.


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2734 E. Natiele Tiago da Silva et al.
The use of pencil graphite electrodes (PGEs) in electro-
chemistry already was previously reported [21–23]. PGE has
chemical properties similar to other carbon-based electrodic
materials in addition to large availability, low cost, easiness of
modification and mechanical robustness [24]. Furthermore,
the surface of the PGEs can be renewed more easily than
the surface of solid electrodes, which usually requires hard-
working polishing procedures [25].
In the field of microfluidics, the use of commercial
graphite pencil leads for electrochemical sensing covers some
few reports that describe the utilization of pencil drawn
electrodes for detection in paper-based devices. Dossi and
co-workers proposed, for the first time, the use of pencil-
drawn electrodes for amperometric detection in ME. The
three-electrode cell developed was obtained through hand-
drawing the electrodes in paper-based fluidic devices using
commercial graphite leads. This system was used for the
separation and detection of ascorbic acid and sunset yellow.
The limits of detection achieved were 30 ␮M for ascorbic
acid and 90 ␮M for sunset yellow [26]. Dossi and co-workers
also described the use of dual pencil-drawn electrode detector
coupled to paper-based fluidic devices for the simultaneous
detection of co-migrating species. The device was used for
the detection of samples containing dopamine and ascorbic
acid and paracetamol and ascorbic acid [27]. Chagas and co-
workers reported, for the first time, the fabrication of pencil
drawn electrodes on paper platforms for capacitively coupled
contactless conductivity detection (C4 D). The electrodes pro-
duced were coupled to PMMA microchips and the system
was used for the electrophoretic separation and detection of
K+ , Na+ and Li+ [2]. Doped pencil leads utilized for drawing
electrodes on paper-based electrochemical devices were also
reported [28–30]. However, to the best of our knowledge, the
use of bare commercial pencil graphite leads as amperomet-
ric sensors in microchip electrophoresis devices has not been
reported yet.
At the same time, nitrite ion is a potentially toxic species
largely used as food preservative. Nitrite may also play the
role of indicator of pollution in waters and environment. The
ingestion of drinking waters contaminated with high levels of
nitrite may lead humans to serious health problems such as
methemoglobinemia and stomach cancer. Thus, monitoring
of the levels of nitrite is an important parameter to preserve
the quality of the water resources [31, 32].
In this context, this work describes, for the first time,
the use of commercial graphite pencil leads as simple elec-
trochemical sensors for amperometric detection in microflu-
idic devices. A PDMS support was fabricated through soft
lithography to accommodate the pencil graphite electrode.
The electrochemical sensor was coupled to PDMS microchips
for the electrophoretic experiments. The system proposed
was characterized and its analytical performance was ex-
plored through the separation and detection of nitrite and
ascorbic acid, selected as model analytes for the study. The
proof of concept for the analytical usefulness of this platform
was performed through its application for the analysis of ni-
trite in well water samples. The nitrite ion was successfully

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Electrophoresis 2017, 38, 2733–2740
quantified at levels below those established by the current
legislations.
2 Materials and methods
2.1 Chemicals and solutions
The following chemicals and materials were used as re-
ceived: dibasic and monobasic potassium phosphates were
acquired from Vetec (Duque de Caxias, RJ, Brazil). Ascor-
bic acid and sodium hydroxide were purchased from Sigma-
Aldrich (St. Louis, MO, USA). Cetyltrimethylammonium bro-
mide (CTAB) was supplied by Merck (Whitehouse Station,
NJ, USA). Sodium nitrite was acquired from Dinâmica (Di-
adema, SP, Brazil). Parafilm was purchased from Fischer Sci-
entific (Waltham, MA, USA). PDMS monomer and its curing
agent were ordered from Dow Corning (Elizabethtown, KY,
USA). Colloidal silver glue was supplied by McMaster-Carr
(Elmhurst, IL, USA). Graphite pencil leads (0.7 mm diam-
eter, type 2B), glass plates (3 mm thickness), epoxy glue,
1500-grit sandpaper and copper wires were purchased at lo-
cal stores. All buffers and aqueous solutions were prepared
using 18.2 M⍀.cm deionized water produced through a Mil-
lipore system (Kansas City, MO, USA). Stock solutions of the
buffer (50 mM) and surfactant (1 mM) were prepared weekly.
The standard solutions of the analytes used for the experi-
ments were prepared daily just through solubilization of the
reagents in ultrapure water.
2.2 Fabrication of the PDMS microchips
The fabrication of the silicon mold used for prototyping the
PDMS microfluidic devices was based on a well-established
method and performed according to a protocol described else-
where [33–35]. The single-T PDMS microchips used in this
work were fabricated through mixing the PDMS monomer
and its curing agent at 9:1 ratio (w/w) and then depositing the
polymer mixture over the silicon mold. The PDMS was cured
at 70°C for 3 h and peeled off from the mold after this time.
The PDMS microchips consisted of 5 cm long separation
channel (from the intersection to the end of the separation
channel) and 0.75 cm long side arms. The depth and width of
the channels were 15 ␮m and 40 ␮m, respectively. The holes
for the reservoirs were drilled using 4 mm and 6 mm biopsy
punches. Fig. S1 (ESI, Electronic Supporting Information)
shows a schematic of the microchip used in this work.
2.3 Fabrication of the support for the working
electrode
The device used as support for the pencil graphite working
electrode (PGWE) was produced as illustrated in Fig. 1: firstly,
a PDMS device containing one channel with the dimensions
of ca. 500 ␮m depth and 500 ␮m height was fabricated using
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Electrophoresis 2017, 38, 2733–2740
Figure 1. Graphical representation of the steps for assembly of
the pencil graphite working electrode: (I) PDMS support contain-
ing the channel for insertion of the pencil graphite lead; (II) PDMS
device reversibly sealed over the glass plate; (III) insertion of the
graphite lead (type 2B, 0.7 mm diameter) previously sanded with
1500-grit sandpaper; (IV) attachment of a piece of copper wire us-
ing epoxy resin for external connection with the potentiostat and
application of colloidal silver glue for electrical contact between
the pencil graphite electrode and the copper wire; (V) alignment
of the PDMS microchip over the pencil graphite working elec-
trode.
soft lithography (I) [19]. The dimensions of this channel were
adjusted to be slightly smaller than the dimensions of the pen-
cil graphite lead used as working electrode. Next, this PDMS
device was reversibly sealed against a glass plate (3 mm thick-
ness × 110 mm length × 95 mm width) in order to give rigid-
ity to the system (II). A pencil graphite lead (0.7 mm diameter,
type 2B) was carefully sanded using 1500-grit sandpaper aim-
ing to obtain a semicircle format. This sanded graphite lead
was manually inserted into the channel on the PDMS device
(III) in a way that the straight part of the graphite lead was
oriented to the top and leveled with the PDMS surface, such
as showed in Fig. S2 (ESI, Electronic Supporting Informa-
tion). In order to ensure the reproducibility of the electrodes,
the graphite pencil leads were always sanded until they were
aligned with the topside of the PDMS device. Due to the fact
that the dimensions of the channel were smaller than the
dimensions of the graphite leads and also due to the elas-
tic properties of PDMS, the channel “involves” the graphite
lead and adjust itself to the shape of the PGWE. A copper
wire was attached to the glass plate with epoxy resin, near to
the PGWE, for the external connections. The electrical con-
tact between the PGWE and the copper wire was achieved
using colloidal silver glue (IV). Finally, the PDMS microchip
was aligned over this device in end-channel configuration (V).
Fig. S3A (ESI, Electronic Supporting Information) shows one
picture of the PDMS support with the PGWE inserted inside
the channel.
2.4 Alignment between the PDMS microchip and
the PGWE
The PDMS microchips were manually positioned and re-
versibly sealed against the PDMS device containing the
graphite pencil working electrode over a microscope. The
electrode was aligned ca. 15–20 ␮m from the end of the
separation channel for all the experiments (end-channel
configuration). Fig. S3B (ESI, Electronic Supporting

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Miniaturization 2735
Information) exhibits one picture of the microchip-electrode
system and Fig. S3C shows one micrograph of the alignment.
2.5 Electrophoresis procedure
Prior to the use, the PDMS microchips were conditioned
under vacuum with 0.1 M NaOH and running buffer dur-
ing 10 min each. The running buffer consisted of 5 mM
phosphate at pH 7.5 containing 200 ␮M CTAB surfactant,
employed to reverse the EOF direction [12].
2.6 Instrumental apparatus
The electrophoretic experiments were performed on a home-
made instrument described elsewhere [12, 18, 19]. Briefly,
it is equipped with a ±2000 VDC high-voltage power sup-
ply (EMCO model CA20N) that provides the separation and
injection voltages to the microchip. A home-made relay-
based device was employed to automatically switch the high
voltages between injection and separation. All experiments
were performed using floating injection. An Autolab po-
tentiostat/galvanostat (model PGSTAT128N) equipped with
extreme low current (ECD) module was used for amper-
ometric detection (AD) and cyclic voltammetry (CV) ex-
periments. The voltammetric and electrophoretic experi-
ments were performed using conventional three-electrode
system composed by the pencil graphite working elec-
trode, commercial Ag/AgCl reference electrode (filled with
3 M KCl) and platinum wire, that served as counter elec-
trode. The detection reservoir of the microchip was used
as electrochemical cell. Fig. S4 (ESI, Electronic Support-
ing Information) shows pictures of the microchip-electrode
platform with the connections such as used during the
experiments.
2.7 Well water sample
The well water used as sample was collected in the rural
area of the city of Campo Grande, MS, Brazil. It was spiked
with known concentrations of nitrite standard since the un-
spiked well water did not show any detectable concentration
of nitrite. The spiked sample was diluted in concentrated run-
ning buffer (50 mM phosphate, pH = 7.5) containing 2 mM
CTAB at 9:1 ratio (v/v) aiming to achieve the final concen-
tration of 5 mM of buffer and 200 ␮M of surfactant. Before
injection into the microchip the diluted sample was filtered
through 0.45 ␮m-membrane. No other pre-treatment was
necessary.
2.8 Scanning electron microscopy (SEM)
The surface of the PGWE was evaluated through SEM tech-
nique. A JSM-6380LV instrument (Jeol, Tokyo, Japan) was
used to obtain images in different magnifications.
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2736 E. Natiele Tiago da Silva et al.
Figure 2. Images of the surface of the PGWE obtained through
SEM before the sanding step (images A and B) and after the
sanding step (images C and D).
3 Results and discussion
3.1 Morphological investigation and electrochemical
behavior of the PGWE
SEM technique was used to evaluate the morphology of
the PGWE. Figure 2 presents SEM images of the pencil
graphite electrode recorded before (Figs. 2A and 2B) and after
(Figs. 2C and 2D) the sanding step. It can be clearly seen that
the sanding changes the shape of the surface of the graphite
pencil lead from circular to flat form. However, the sanding
does not affect significantly the morphology of the PGWE.
The sanded surface showed itself as slightly rougher than
the surface of the PGWE before sanding, but both structures
presented themselves as quite similar.
The electrochemical activity of the PGWE was initially
evaluated through recording cyclic voltammograms of two se-
lected analytes. The electrochemical oxidation of nitrite and
ascorbic acid was monitored in an optimized aqueous media
composed of 5 mM phosphate buffer at pH 7.5 with 200 ␮M
CTAB surfactant. Both substances presented electrochemical
response over the surface of the PGWE, as can be observed
in Figure S5 (ESI, Electronic Supporting Information). Ni-
trite and ascorbate showed oxidation peaks at Ep = +0.85 V
and Ep = +0.58 V, respectively. It confirms the electrochem-
ical activity of the graphite electrode for the detection of the
selected analytes.
3.2 Electrophoretic experiments
The performance of the graphite pencil electrode as ampero-
metric sensor for microchip electrophoresis was investigated
through the coupling of the PGWE to PDMS microchips
in end-channel configuration. This system was employed
to monitor the electrophoretic separation of the probe an-
alytes (nitrite and ascorbate). Experimental conditions such

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Electrophoresis 2017, 38, 2733–2740
as buffer, pH and voltages of injection and separation were
studied and optimized in order to obtain the best conditions
for the analysis. The best results regarding the peak resolu-
tion, analytical sensitivity, peak broadening and analysis time
were achieved when a medium composed of 5 mM phos-
phate buffer at the pH value of 7.5 was used. The use of a
cationic surfactant was necessary to reverse the EOF direc-
tion since the selected analytes are in their anionic form at
the optimized pH and because of this the separations had to
be carried out using reverse polarity. For this purpose, the
cationic surfactant CTAB was added to the running buffer
at the optimized concentration of 200 ␮M [12, 18]. The volt-
ages of separation, injection and the time of injection were
optimized to be −1000 V, −600 V, and 5 s, respectively.
The PGWE was used to obtain hydrodynamic voltammo-
grams for the analytes, such as depicted in Fig. 3A. The effect
of the detection potential on the analytical signal of nitrite and
ascorbate was investigated in the range from +0.5 V to +1.2 V
(vs. Ag/AgCl), on increments of +0.1 V. As can be observed,
nitrite presented the highest peak current at +1.0 V and ascor-
bate showed its maximum response at +0.9 V. Aiming to
obtain stable background current and good signal/noise ratio
for both analytes, the optimum detection potential adopted
was +1.0 V.
Figure 3B presents one electropherogram of nitrite and
ascorbate recorded under the optimized conditions. The com-
pounds were successfully separated and detected using the
PGWE in less than 60 s. The baseline and the peak profiles ob-
served were very satisfactory. Sensitivities of ca. 196 pA/␮M
and ca. 93 pA/␮M were obtained from individual measure-
ments of nitrite and ascorbate, respectively. The value calcu-
lated for the background noise was ca. 200 pA.
The PDMS microchip coupled to the PGWE provided
acceptable values for parameters such as peak resolution
and separation efficiency regarding the analytes selected.
Using the optimized experimental conditions, the value of
1.0 was achieved for the peak resolution. Under the same
conditions, separation efficiencies of ca. 3500 plates/m and
ca. 2400 plates/m were obtained for nitrite and ascorbate,
respectively.
3.3 Evaluation of the stability of the system
The repeatability of the PGWE was investigated through car-
rying out successive measurements of the selected analytes
using the system developed. Fig. S6 (ESI, Electronic Sup-
porting Information) presents one electropherogram con-
taining ten successive injections of nitrite and ascorbate.
As can be observed, the analytical signal remained stable
for both analytes. RSD values of 1.6 and 12.3%, regarding
the peak currents, were found for nitrite and ascorbate, re-
spectively (for n = 10 measurements). The high value of
RSD observed for ascorbic acid may be attributed to the
well-known characteristic of this substance and of its oxi-
dation products to adsorb and foul the electrode surface [36].
This effect may also affect the analytical signal of nitrite.
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Electrophoresis 2017, 38, 2733–2740 Miniaturization 2737

3.4 Construction of the analytical curve

In order to determine the linear concentration ranges of

the analytes over the PGWE, analytical curves were con-
structed through injection of standards of nitrite and ascor-
bate in divers concentrations. Figure 4 exhibits the cali-
bration curves obtained in the range from 10 to 200 ␮M
for nitrite and from 20 to 400 ␮M for ascorbate. Both
curves presented linear dependence for the concentration
ranges evaluated. The linear equations calculated were
Ip /nA = 3.33 ± 0.15 + 0.16 ± 0.01 × [nitrite]/␮M for nitrite
and Ip /nA = 2.78 ± 0.09 + 0.047 ± 0.002 × [ascorbate]/␮M

Figure 3. (A) hydrodynamic voltammograms of nitrite and ascor-

bate recorded in different potentials of detection (results for n =
3 measurements); (B) electrophogram recorded at the optimized
potential of detection of +1.0 V (versus Ag/AgCl) for a mixture
of 300 ␮M nitrite and 500 ␮M ascorbate. Other conditions: 5 mM
phosphate buffer at pH value of 7.5 containing 200 ␮M CTAB;
separation voltage: −1000 V; injection voltage: −600 V; injection
time: 5 s.

However, due to the large availability and cheapness of the

pencil graphite leads, this inconvenience can be easily fig-
ured out simply through replacement of the fouled PGWE
for a new one.
The reproducibility was investigated by monitoring the
electrophoretic separation of the analytes under the optimized
conditions using four different pencil graphite working elec-
trodes. The values found for the RSD, regarding the analytical
signal, were 3.7% for nitrite and 1.6% for ascorbate.
The durability of the PGWE also was evaluated. The
lifetime of each PGWE was estimated to be ca. 700 elec-
trophoretic runs. Based on these data, the PGWE showed
acceptable stability to be used as electrochemical sensor for
detection of target analytes on microchip electrophoresis.
Figure 4. (A) electropherograms of nitrite and ascorbate in differ-
ent levels of concentration: (a) 10 ␮M nitrite and 20 ␮M ascorbic
acid; (b) 20 ␮M nitrite and 40 ␮M ascorbic acid; (c) 40 ␮M nitrite
and 80 ␮M ascorbic acid; d) 80 ␮M nitrite and 160 ␮M ascorbic
acid; (e) 120 ␮M nitrite and 240 ␮M ascorbic acid; (f) 180 ␮M nitrite
and 320 ␮M ascorbic acid and (g) 200 ␮M nitrite and 400 ␮M ascor-
bic acid. (B) analytical curve of nitrite and (C) analytical curve of
ascorbate (results for n = 3 measurements). Conditions: potential
of detection: +1.0 V; other conditions: see Fig. 3.


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2738 E. Natiele Tiago da Silva et al. Electrophoresis 2017, 38, 2733–2740

Table 1. Comparison between the limits of detection obtained with the PDMS-PGWE system proposed here and some similar works that
also employ microfluidic devices coupled to amperometric detection for the analysis of nitrite and/or ascorbate

System LOD/␮M

Nitrite Ascorbate Reference Year

Glass-Cu-CPEa) 7.6 N.A. [38] 2009

PDMS-Ptb) 6.2 N.A. [33] 2011
PDMS-CFEc) N.A. 9.7 [19] 2014
SU-8/Pyrex-Ptd) N.A. 7.3 [12] 2015
PDMS-CIEe) 8.2 12.7 [18] 2017
PDMS-PGWEf) 2.8 5.7 This work -

a) Glass microchips coupled to copper (3-mercaptopropyl)trimethoxysilane [Cu(II)-MPS] complex-modified carbon paste electrode.
b) PDMS microchips coupled to platinum electrode.
c) PDMS microchips coupled to carbon fiber electrode.
d) SU-8/Pyrex microchip with integrated platinum electrode.
e) PDMS microchips coupled to carbon-ink electrode.
f) PDMS microchips coupled to pencil graphite working electrode.
N.A. = not applicable

for ascorbate. The two curves exhibited the value of 0.996 for electropherograms recorded during the determinations in the
the correlation coefficient (R). samples are depicted in Fig. 5. Nitrite was successfully de-
The values for the limits of detection (LOD) and quantifi- tected in the samples with the PGWE at levels below its MCL
cation (LOQ) were determined using the relationship 3SDa /b and the results of the analyses presented good agreement
and 10SDa /b, respectively, where SDa is the standard devia- when compared with the reference values (RSD ranging from
tion of the curve intercept and b is the slope of the analytical 2.2 to 7.2%). Moreover, no interference of other ions/species
curve. The LODs found were 2.8 ␮M for nitrite and 5.7 ␮M for likely present in the sample were observed since the sig-
ascorbate. The limits of detection achieved here were lower nal/noise ratio was satisfactory and the migration times and
than those presented in previous works that describe the use analytical signal remained stable. These data confirm the an-
of microchip electrophoresis coupled to amperometric detec- alytical feasibility on the use of the PGWE as amperometric
tion in end-channel configuration for the detection of nitrite sensor in microchip electrophoresis and on the use of this
and ascorbate. Table 1 displays a comparison between these microfluidic system for the determination of target analytes
values.
Table 2 summarizes some parameters calculated for the
system evaluated. According to the data achieved, the ana-
lytical performance of the PDMS microchip-PGWE platform
Table 2. Parameters calculated for the electrophoretic separation
can certainly be considered as a promising option for ampero- of nitrite and ascorbate using PDMS microchips coupled
metric detection in microchip electrophoresis devices. Other to the PGWE
benefits that may be pondered regarding the use of PGWE for
electrochemical detection in ME are the large availability of Nitrite Ascorbate
the material used in conjunction with its satisfactory lifetime Migration time (tm /s) 24.1 39.0
and low cost (ca. $ 0.05 per pencil graphite lead). Repeatability intra-electrode 1.6 12.3
Ip a) /%RSD
Repeatability intra-electrode 2.5 2.8
tm a) /%RSD
3.5 Analysis of real samples
Reproducibility inter-electrodes 3.7 1.6
Ip b) /%RSD
The usefulness of the proposed microfluidic platform for real Reproducibility inter-electrodes 1.8 0.26
applications was tested through carrying out the determina- tm b) /%RSD
tion of nitrite in well water samples. The maximum contam- Theoretical plates (N) 172 118
inant level (MCL) determined by Brazil and US legislations Efficiency /plates m−1 3,470 ± 170 2,364 ± 110
for nitrite in natural waters is 1.0 mg/L (21.7 ␮M) [37]. Resolution between the peaks 1.01
The well water samples were spiked with nitrite stan- Linear concentration range / ␮M 10 to 200 20 to 400
dard in two different levels of concentration before injection Sensitivity / pA ␮M−1 196 93
in the microchip. The analytical assays were performed us- LOD / ␮M 2.8 5.7
LOQ / ␮M 9.4 18.8
ing the standard addition method aiming to avoid matrix
effects. In order to check the precision of the measurements, Results for a) n = 10 measurements and
the quantifications were carried out in triplicate. Some typical b) n = 4 different working electrodes.


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Electrophoresis 2017, 38, 2733–2740 Miniaturization 2739

compounds usually present in natural waters that may af-

fect the analysis. Concentrations of 200 ␮M each of Zn2+ ,
Mn2+ , Fe2+ , Cu2+ , Co2+ , Ni2+ , Pb2+ , Cd2+ , Hg2+ , As3+ and
NH4 + were added to the spiked well water sample. No sig-
nificant interference of these species was observed during
the analyses and nitrite was successfully quantified. The sig-
nal/noise ratio, analytical signal and migration times did
not present meaningful changes. The recovery rates of ni-
trite ranged from 97 to 105% in the presence of the species
added.

4 Concluding remarks

Figure 5. (A) electropherograms recorded during one of the de-
terminations carried out in the well water sample spiked with
nitrite. (B) respective standard addition curve constructed. Re-
sults for n = 3 measurements. Experimental conditions: see
Fig. 4.
in routine analyses. Table 3 exhibits in details the results
achieved during the determinations of nitrite in the samples.
3.6 Evaluation of possible interferings
The selectivity of the system proposed was evaluated through
carrying out the determination of nitrite in the presence of
We reported in this work, for the first time, the use of in-
expensive graphite pencil leads as working electrodes for
amperometric detection in microchip electrophoresis. The
graphite electrode was inserted into a channel fabricated on a
PDMS device used for assembly the electrode. The morphol-
ogy and the electrochemical activity of the pencil graphite
electrode were investigated. SEM experiments showed that
the morphology of the working electrode was not affected
by the sanding step. Furthermore, cyclic voltammetry experi-
ments confirmed the electrochemical activity of the electrode
through the oxidation of nitrite and ascorbic acid that were
chosen as model analytes for the study.
The pencil graphite electrode was coupled to PDMS mi-
crochips in end-channel configuration to monitor the elec-
trophoretic separation of the selected analytes. The separa-
tion and detection of nitrite and ascorbate were successfully
performed using this microfluidic system and the related ex-
perimental parameters were optimized in order to achieve
the best conditions for the analysis. The graphite electrode
presented satisfactory analytical performance in conjunction
with good repeatability and reproducibility. As proof of prin-
ciple of its analytical feasibility, the sensor proposed here was
effectively employed for the determination of nitrite in real
well water samples at levels below the MCL established by
Brazil and US.
Considering the large availability and the low cost of the
material employed (ca. $ 0.05 per piece) and based on the
data reported here, the use of pencil graphite leads for de-
tection on ME presents itself as a very promising alternative
for the rapid and inexpensive production of amperometric
sensors for microfluidic devices. Moreover, the approach de-
scribed here opens new possibilities for applications such

Table 3. Results obtained for the determinations of nitrite in the well water samples using the microfluidic system proposed

Nitrite spiking/␮M Nitrite found/␮M Recovery/% Average/␮M±SD RSD/%

12.8 102.4
12.5 12.2 97.6 12.4 ± 0.3 2.6
12.3 98.4
25.0 25.3 101.2
28.6 114.4 26.4 ± 1.9 7.1
25.4 101.6


C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
2740 E. Natiele Tiago da Silva et al.
as the fabrication of systems containing dual-electrodes, de-
couplers for in-channel configuration or even graphite-based
modified electrodes aiming the enhancement of the analytical
sensitivity.
This work was funded by Conselho Nacional de Desenvolvi-
mento Cientı́fico e Tecnológico (CNPq) - grant No. 485614/2012-
0. The authors also acknowledge Coordenação de Aperfeiçoamento
de Pessoal de Nı́vel Superior (CAPES) for the scholarships
granted to Eiva Natiele Tiago da Silva and Jacqueline Mar-
ques Petroni and CNPq for the researcher fellowship granted
to Valdir Souza Ferreira. Lastly, the authors also would like
to thank the Laboratory of Microfabrication (LMF) from the
Brazilian Nanotechnology National Laboratory (LNNano) and
the Multiuser Laboratory for Analysis of Materials (MULTI-
LAM) from the Physics Institute (INFI) of the Federal University
of Mato Grosso do Sul (UFMS) for using their facilities.
The authors have declared no conflict of interest.
5 References
[1] Chen, C. H., Lin, Y. T., Lin, M. S., Anal. Chim. Acta 2015,
874, 33–39.
[2] Chagas, C. L. S., Costa Duarte, L., Lobo-Júnior, E. O., Pic-
cin, E., Dossi, N., Coltro, W. K. T., Electrophoresis 2015,
36, 1837–1844.
[3] Gomez, F. J. V., Martı́n, A., Silva, M. F., Escarpa, A., Elec-
trophoresis 2015, 36, 1880–1885.
[4] Shang, F., Guihen, E., Glennon, J. D., Electrophoresis
2012, 33, 105–116.
[5] Am, J., Zhiwei, Z., Kang Kug, L., Chong, H. A., Paul, L.
B., Meas. Sci. Technol. 2011, 22, 1–18.
[6] Martin, A., Vilela, D., Escarpa, A., Electrophoresis 2012,
33, 2212–2227.
[7] Ho, C. M. B., Ng, S. H., Li, K. H. H., Yoon, Y.-J., Lab Chip
2015, 15, 3627–3637.
[8] Temiz, Y., Lovchik, R. D., Kaigala, G. V., Delamarche, E.,
Microelectron. Eng. 2015, 132, 156–175.
[9] Pumera, M., Electrophoresis 2008, 29, 269–273.
[10] Gabriel, E. F. M., Duarte Junior, G. F., Garcia, P. de T., de
Jesus, D. P., Coltro, W. K. T., Electrophoresis 2012, 33,
2660–2667.
[11] Saylor, R. A., Reid, E. A., Lunte, S. M., Electrophoresis
2015, 36, 1912–1919.
[12] Lucca, B. G., de Lima, F., Coltro, W. K. T., Ferreira, V. S.,
Electrophoresis 2015, 36, 1886–1893.
[13] Lee, H. L., Chen, S. C., Talanta 2004, 64, 210–216.
[14] Pozo-Ayuso, D. F., Castano-Alvarez, M., Fernandez-la-
Villa, A., Garcia-Granda, M., Fernandez-Abedul, M. T.,
Costa-Garcia, A., Rodriguez-Garcia, J., J. Chromatogr. A
2008, 1180, 193–202.

C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2017, 38, 2733–2740
[15] Martin, A., Lopez, M. A., Gonzalez, M. C., Escarpa, A.,
Electrophoresis 2015, 36, 179–194.
[16] Chicharro, M., Sánchez, A., Bermejo, E., Zapardiel, A.,
Rubianes, M. D., Rivas, G. A., Anal. Chim. Acta 2005,
543, 84–91.
[17] Xu, J., Zhang, L., Chen, G., Electrophoresis 2013, 34,
2017–2024.
[18] Petroni, J. M., Lucca, B. G., Ferreira, V. S., Anal. Chim.
Acta 2017, 954, 88–96.
[19] Lucca, B. G., Lunte, S. M., Coltro, W. K. T., Ferreira, V. S.,
Electrophoresis 2014, 35, 3363–3370.
[20] Regel, A., Lunte, S., Electrophoresis 2013, 34, 2101–2106.
[21] Akanda, M. R., Sohail, M., Aziz, M. A., Kawde, A.-N.,
Electroanalysis 2016, 28, 408–424.
[22] Kawde, A.-N., Baig, N., Sajid, M., RSC Adv. 2016, 6,
91325–91340.
[23] David, I. G., Popa, D.-E., Buleandra, M., J. Anal. Methods
Chem. 2017, 2017, 1–22.
[24] Levent, A., Yardim, Y., Senturk, Z., Electrochim. Acta
2009, 55, 190–195.
[25] Wang, J., Kawde, A.-N., Sahlin, E., Analyst 2000, 125,
5–7.
[26] Dossi, N., Toniolo, R., Pizzariello, A., Impellizzieri, F.,
Piccin, E., Bontempelli, G., Electrophoresis 2013, 34,
2085–2091.
[27] Dossi, N., Toniolo, R., Piccin, E., Susmel, S., Pizzariello,
A., Bontempelli, G., Electroanalysis 2013, 25, 2515–
2522.
[28] Dossi, N., Toniolo, R., Impellizzieri, F., Bontempelli, G., J.
Electroanal. Chem. 2014, 722–723, 90–94.
[29] Dossi, N., Toniolo, R., Terzi, F., Impellizzieri, F.,
Bontempelli, G., Electrochim. Acta 2014, 146, 518–
524.
[30] Dossi, N., Toniolo, R., Terzi, F., Piccin, E., Bontempelli, G.,
Electrophoresis 2015, 36, 1830–1836.
[31] Zhu, N., Xu, Q., Li, S., Gao, H., Electrochem. Commun.
2009, 11, 2308–2311.
[32] Mehmeti, E., Stankovic, D. M., Hajrizi, A., Kalcher, K.,
Talanta 2016, 159, 34–39.
[33] Gunasekara, D. B., Hulvey, M. K., Lunte, S. M., Elec-
trophoresis 2011, 32, 832–837.
[34] Martin, R. S., Gawron, A. J., Lunte, S. M., Anal. Chem.
2000, 72, 3196–3202.
[35] Meneses, D., Gunasekara, D. B., Pichetsurnthorn, P., da
Silva, J. A. F., de Abreu, F. C., Lunte, S. M., Electrophore-
sis 2015, 36, 441–448.
[36] Huang, J., Liu, Y., Hou, H., You, T., Biosens. Bioelectron.
2008, 24, 632–637.
[37] Shariati-Rad, M., Irandoust, M., Mohammadi, S., Spec-
trochim. Acta A Mol. Biomol. Spectrosc. 2015, 149,
190–195.
[38] Shiddiky, M. J., Lee, K. S., Son, J., Park, D. S., Shim, Y.
B., J. Agric. Food Chem. 2009, 57, 4051–4057.
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