Electrochimica Acta 283 (2018) 1498e1506

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Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta

Single frequency impedance strategy employed in rapid detection of

leukemia cancer cells using an electrospun PES-nanofiber reinforced
ternary composite-based cytosensor
Mojtaba Shamsipur a, *, Mohammad Bagher Gholivand a, Hosna Ehzari a,
Afshin Pashabadi a, Elham Arkan b, Kamran Mansouri c
a
Department of Chemistry, Razi University, Kermanshah, Iran
b
Nano Drug Delivery Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
c
Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Cancer is one of the main causes of death in today's world, therefore, extending fast and reliable ap-
Received 26 April 2018 proaches for early monitoring of cancer cells have become one of the great challenges for scientists. This
Received in revised form work reports exploiting a fast single frequency impedance strategy coupled to a new constructed
14 July 2018
composite for the quantitative detection of the human chronic myelogenous leukemia (CML) cell lines,
Accepted 15 July 2018
Available online 18 July 2018
K562. The platform used for the fabrication of the cytosensor is based on an electrospun poly[ether-
sulfone] (PES) sub-layer ready for improving by the upper nanodimension layers comprised of gold
nanoparticles and multiwall carbon nanotubes (AuNPs/MWCNTs). The binding of K562 cancer cells with
Keywords:
Leukemia K562 cancer cells
the pre-immobilized capture ssDNA was successfully transduced by Faradaic impedance spectroscopy
Electrospun nano fibers (FIS). To diminish the possible nonspecific impedance changes that may cause some uncertainties in FIS
Faradic impedance spectroscopy signal, and to considerably decrease the time of the extended method, a fast single frequency mea-
Single frequency measurement surement (SFM) strategy was tested based on recording total impedance jZj in different individual fre-
quencies, the methodology that was succeeded to recognize the target cells in measurement times as low
as several seconds. Under the optimum conditions, the assessed signals were proportional to the pop-
ulation of K562 cells from 102 to 107 cells mL1 with a calculated detection limit of 60 cells mL1. The
proposed biosensing-device also demonstrated high stability and reproducibility, which was able to offer
great promise for the quantitative assay of K562 in routine analyses. The results of real sample analyses
introduced the proposed device as alternative tool for CML K562 cell detection in real biological samples.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction
With the rise of cancer threat that causes innumerable world-
wide cancer fatality, its early diagnosis and advance in treatment
have received great attention [1,2]. Chronic myelogenous leukemia
(CML), a malignancy of pluripotent hematopoietic cells, is a type of
cancer that starts in certain blood-forming cells of the bone
marrow. It cause proliferation of mature granulocytes (neutrophils,
eosinophils and basophils) and their precursors. K562, as one of the
most aggressive human chronic myelogenous leukemia (CML) cell
lines, is derived from CML patients, therefore its early detection via
* Corresponding author.
E-mail address: mshamsipur@yahoo.com (M. Shamsipur).
a rapid and reliable method will be a great help to these patients
[3,4].
The conventional detection methods for K562 are the cyto-
chemistry test [5], the bone marrow consideration [6], the immu-
nophenotyping [7] and the bone marrow minimal residual disease
examination [8]. However, almost all these methods faced the
problems of low specificity, high testing costs, sophisticated
instrumentation, time-consuming procedure, and need to a bone
marrow examination that cause severe pain for the patients [9].
These severities can deter early-stage patients from diagnostic tests
and reduce their chance to exploit point-of-care tools, the occur-
rence that can be very dangerous.
In the recent decade, a genetic abnormalities detection meth-
odology has attracted a great interest [10,11] for diagnostic of CML.
Concurrently, RT-PCR method was introduced for the study of bcr/

https://doi.org/10.1016/j.electacta.2018.07.089
0013-4686/© 2018 Elsevier Ltd. All rights reserved.
 M. Shamsipur et al. / Electrochimica Acta 283 (2018) 1498e1506
abl fusion gene, as a specific fusion gene of CML [12,13]. Apart from
the benefits, these methods required the complex procedures such
as the extraction and reverses transcription of RNA [14], moreover,
the PCR method may generates large positive or negative un-
certainties in the obtained response.
In the last few years, electrochemical biosensing devices have
been well recognized as a cost effective, easy use, specific and
sensitive detection approach for K562 cells. Among them, the
constructed devices joined with the Faradaic impedance spectros-
copy (FIS) technique have attracted great interests. The combina-
tion of bio-recognition elements with the FIS delivered two most
important advantages, i.e., high sensitivity toward affinity events at
interface of electrode/electrolyte, and transduction of surface
changes through a nondestructive approach. However, these ben-
efits are often associated with some sources of uncertainties
through signal measurements, which are mainly caused by time-
length of either of individual measurement that allows diffusion
of charged probe species into biosensing layer engineered at the
surface of transducer [15e17]. To minimize the sources of un-
certainties, an exclusive solution has been proposed based on using
a measurement approach that decreases possible non-specific
measurement related-changes. Operationally, it will be applied by
limiting the possibility of diffusion of probe species during signal
recording in FIS, which mostly occurs in low frequencies region
(Warburg portion). The aborting the Warburg portion can assist in
decreasing the possible non-specific signal changes. Another
strategy rely on signal assessment by “single frequency measure-
ment” (SFM), the procedure that is able to dramatically reduce the
measurement time to a few seconds and even shorter.
Electrospinning as a versatile method was utilized to fabricate a
polymer-based nanofiber in role of platform to incorporate the
exclusive properties of nanostructure shaped PES in a new
biosensor for fast monitoring of K562. There are various advantages
for the PES, including the resistance to many solvents and chem-
icals, mechanical stability, good electrical characteristics, low creep
and easy processing in sensor construction [18]. The combination of
these advantages with the well-known properties of AuNPs in
electrochemistry aided us to fabricate a platform tailored for bio-
sensing application, where a sensitive device with good long-term
stability and repeatability was established for detection of K562.
During recent decades, frequent reports have expressed different
abilities for AuNPs in electrochemical (bio) sensors. It has proper
biocompatibility in biomolecules detection, high effective surface
area, improved sensitivity and outstanding electrocatalytic activity
[19]. AuNPs function as “electron antennae” that efficiently trans-
port electrons between the electrode and the electroactive species,
thus, promoting the kinetics of electron transfer processes [20,21].
In this work, we introduced a robust and biocompatible ternary
composite employed in immobilization of a capture probe ssDNA
able to assay K562 cells. The poly(ether sulfone) (PES) was
appointed for the construction of an electrospun nanofibers sub-
layer, since it could provide a mechanical strength, continuous
porous structure with a large surface area, chemical and biological
compatibility, and proper electrical conductivity [18,22,23]. The
results of comparative examinations disclosed the best perfor-
mance for the ternary composite PES/AuNPs/MWCN in trans-
duction of the tiny specific binding events occurred between the
recognition element, ssDNA, and the K562 cancer cells target at the
electrode/electrolyte interface by FIS. Exploiting SFM in different
individual frequencies makes it possible to transduce the occurred
affinity interactions over times around a few seconds, depending
the magnitude of the applied frequency. It can minimize possible
sources of uncertainties caused by non-specific interactions during
relatively long measurement time for recording Nyquist plots.
Finally, the obtained data with SFM were compared to the main
1499
calibration curve, assessed by the multiple individual frequency
procedure, and the results displayed a promising feature for the
established biosensing strategy based on SFM.
2. Experimental
2.1. Reagents and chemicals
All chemicals were of analytical reagent grade and used without
further purification. N-(3-dimethylaminopropyl)-N0 -ethyl-
carbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide
(NHS, 98%) were obtained from SigmaeAldrich (St. Louis, MO, USA),
[K3Fe(CN)6] and [K4Fe(CN)6] were obtained from Merck (Darm-
stadt, Germany). HAuCl4.3H2O were obtained from Shanghai Re-
agent Company (Shanghai, China). MWCNTs were purchased from
Nanoport. Co. Ltd. (Shenzhen, China). Bovine serum albumin (BSA)
was obtained from SigmaeAldrich Chemical Co. (USA). The
aptamer [5ˊ-(NH2)-TTT TTT TTT TAC AGC AGA TCA GTC TAT CTT CTC
CTG ATG GGT TCC TAT TTA TAG GTG AAG CTG T-3ˊ was synthesized
and purified by Faza Biotech Co. Ltd (Tehran, Iran).
2.2. Culture and collection of cells
The leukemia K562 cell line was cultured in RPMI and supple-
mented with 10% fetal bovine serum, penicillin (0.1 g mL1), and
streptomycin (0.1 g mL1) in an incubator (5% CO2, 37  C). After
collecting, the cells sample were centrifuged at 500 rpm (5 min)
and separated from medium, it was washed twice with sterilized
0.01 M PBS (pH 7.4). The sediment was further suspended in spe-
cific volume of 0.1 M of PBS buffer to obtain final concentration of
107 cell mL1 (stock solution). The desired concentration was ob-
tained by successive dilution from the stock solution. The cell
numbers were estimated by a Petroff-Hausser cell counter.
2.3. Apparatus and measurements
Electrochemical measurements were performed with a m-
Autolab type III (Eco Chemie B.V.A) computer-controlled poten-
tiostat and running with the Nova 1.11 software. The electro-
chemical cell was equipped with a saturated Ag/AgCl as reference
electrode, a Pt wire as auxiliary electrode, and with the prepared
biosensor as the working electrodes. FIS measurements were
possessed in a solution containing K3Fe(CN)6/K4Fe(CN)6 (1:1,
0.5 mM) as the redox probe, with an AC voltage amplitude of 5 mV
and a frequency range of 10 kHz to 0.01 Hz at an open-circuit po-
tential (OCP). For the fabrication of PES-NF, a laboratory scale
electrospinning setup was employed.
2.4. Preparation of GCE/PES-NF/AuNPs/MWCNT/ssDNA
After the electrode cleaning with commonly used procedures,
the GCE/NF was prepared by electrospinning in the solution of PES
(20 wt %) in ethanol. At first, the solution of 3% wt. of PES was
prepared in ethanol and sonicated for 30 min to disperse it
completely. The electrospinning process was performed under
electric fields of ca. 100 kV m1. The voltage applied to a 30 cm gap
between the spinneret and the collector (glass carbon electrode).
Next, the AuNPs were electrodeposited onto the GCE/NF by CV
scanning from 0.2e1.2 V in a solution of 0.5 M H2SO4 containing
0.6 M of HAuCl4 at a scan rate of 50 mV s1 for 15 cycles. In the next
step, 1 mg of the treated MWCNTs were dispersed in 1 mL of DMF
by an ultrasonic bath for one hour; then 2 mL of the prepared sus-
pension was dropped onto the surface of the GCE/NF/AuNPs and
was dried at room temperature. For preparing the biosensor, the
GCE/NF/AuNPs/MWCNTs were immersed in 0.05 M PBS (pH 7.0)
1500 M. Shamsipur et al. / Electrochimica Acta 283 (2018) 1498e1506
containing 20 mM of EDC and 40 mM of NHS for 30 min to activate
the interfacial carboxylic groups at the surface confined MWCNTs
on the composite platform. In following and after quick washing
with PBS, 2 mL of 10 mM of aptamer was carefully drop-casted onto
the pretreated composite electrode and dried for 1 h in RT. The
fabricated biosensor was soaked in 1% BSA for 30 min to eliminate
the possible nonspecific adsorption. The overall schematic of the
composite electrode preparation and biosensing approach is shown
in Scheme 1. Finally, the GCE/NF/AuNPs/MWCNT/ssDNA was dried
in air and stored at 4  C in 0.1 M PBS of pH 7.4 when not in use.
3. Results and discussion
3.1. Morphological and electrochemical characterizations of the
GCE/PES-NF/AuNPs/MWCNT
Fig. 1 shows the scanning electron microscopy (SEM) image
taken for different exposed layers GCE/PES-NF (A), GCE/PES-NF/
AuNPs/(B) and GCE/PES-NF/AuNPs/MWCNT (C). Fig. 1A illustrates
the two different magnification SEM image taken from GCE/NF that
shows a diameter about 200 ± 50 nm for the electrospun PES
nanofibers. The represented SEM image for GCE/NF/AuNPs (Fig. 1B)
allows for comparison size of the electrodeposited AuNPs that
homogeneously are distributed on the PES-NFs. Further consider-
ation disclosed the size of AuNPs ca. 22 ± 7 nm. The next image
(Fig. 1C) describes the morphology of the surface covered by a well-
dispersed solution of MWCNT. It demonstrates a mean diameter of
about 51 ± 6 nm for the MWCNTs that are fully covered the GCE/
PES-NF/AuNPs (C). The Energy-dispersive X-ray spectroscopy
(EDX) and Scanning Electron Microscopy/Energy Dispersive X-ray
Spectroscopy (SEM/EDX) images are shown in Fig. 1D. The EDX and
EDX/SEM clearly indicates the presence of different percentages of
C, Au, and O at the prepared composite electrode.
To illuminate the capability of interfacial charge transfer for the
different exposed layers, FIS and CV were carried out in a solution of
0.5 mM [Fe(CN)6]3-/4- and 0.1 M KCl. The corresponding Nyquist
plots (Fig. 2A) and voltammograms (Fig. 2B) are represented for
Scheme 1. Overall illustration for fabrication of the K562 biosensor.
GCE (a), GCE/NF (b), GCE/NF/AuNPs (c) and GCE/NF/AuNPs/
MWCNTs (d). In order to avoid the possible “measurement related
impedance changes” with higher concentrations of charged probe
[16,24], we performed measurements using 0.5 mM of [Fe(CN)6]3-/
4-
(instead of the typically used concentrations 5e10 mM). Fig. 2A
displays the Nyquist plots recorded in 0.5 mM of the redox probe.
An ideal Nyquist plot demonstrates a semicircle with a diameter
corresponding to the charge transfer resistance (Rct) of heteroge-
neous redox reaction of the probe's Faradaic process at the elec-
trode/electrolyte interface. In a usual manner, the semicircle is
followed by diagonal straight line, emerged at lower frequencies,
corresponding to the impedance of current due to diffusion elec-
troactive species from solution to the electrode interface (Warburg
element, W). Then, in order to calculate the relevant Rct, the ob-
tained impedance curve was fitted into an electrical circuit, which
was designed based on the features of the obtained impedance
spectrum. The solution resistance (Rs) arises from the finite
conductance of the ions in bulk solution, which is generally not
affected by binding events.
The capacitance between the electrode and array of ions in so-
lution can be modeled as a series of combination of surface
capacitance and double layer capacitance [24e27]. The impedance
spectra of the modified electrode were consistent with a modified
Randles' model, where, the double-layer capacitance (Cdl) is
replaced by a constant phase element (CPE), due to “frequency
dispersion”. The terms frequency dispersion (or capacitance
dispersion) arise from diverse source that heterogeneously
distribute capacitance and is attributed to surface-roughness/-
heterogeneities, fractal geometry caused by dilatational symme-
try fact, porosity of surfaces, various local composition and slow
adsorption reactions [28e30]. The represented Nyquist plot for the
bare GCE displayed a relatively small Rct (Fig. 2A curve a), mean-
while, the Rct value disclosed a great enhancement for the
possessed curve at the GCE/NF (Fig. 2A, curve b), suggesting that
the electrospinning of PES onto the surface of GCE dramatically
disturbs the charge transfer process and capability of charge-
transfer was diminished (Fig. 2A, curve b).
 M. Shamsipur et al. / Electrochimica Acta 283 (2018) 1498e1506
Fig. 1. SEM images of GCE/PES-NF (A), GCE/NF/AuNPs (B), GCE/NF/AuNPs MWCNTs (C). SEM-EDAX and EDAX spectrum recorded at GCE/NF/AuNPs MWCNTs elemental different
showing signal of C, O and Au, respectively, as is illustrated (D).
Importantly, it should be pointed out that over the middle range
of pH in aqueous solutions, the relatively hydrophobic PES surfaces
strongly adsorbs anions and revealed a negative zeta potential
values [31]. Therefore, the electrostatic interaction between the
negative charged GCE/PES-NF and the negatively-charged redox
probe, [Fe(CN)6]3-/4-, can justifies one portion of the significance
impedance increase in Fig. 2A curve b. As expected, the electrically-
driven deposition of AuNPs on the GCE/NF impressively decreased
the diameter of impedance spectra (Fig. 2A, curve c). After the
anchoring of MWCNTs on GCE/NF/AuNPs, the composite electrode
exhibited a much lower interfacial charge transfer resistance for the
occurred redox reaction (Fig. 2A, curve d), providing a well-
established composite surface ready for induction of the appro-
priate selectivity toward K562-cell by the desired sequenced of
anti-K562 aptamer. For further clarification, the corresponding
cyclic voltammograms after each step of surface modification were
recorded and the results found to be in agreement with the
impedance data (Fig. 2B). The preliminary examinations exhibited
1501
that the use of PES-NF as a sublayer could deliver a functionalized
platform with the higher effective surface area. This property,
coupled with the upper nanostructured layers AuNPs and func-
tionalized MWCNTs, assisted us to prepare a device with much
higher density of recognition elements at a particular geometric
area. In addition, the presence of NF as a robust hydrophobic sub-
layer can assist the composite to keep intact the established ar-
chitecture in an entirely hydrophilic region. This feature could
prolong the life-time and stability of the sensing device, the subject
that through the experimental studies was proved.
In the next experiments, the successful immobilization of the
capture probes on the GCE/PES-NF/AuNPs/MWCNT was explored
by FIS and CV (Fig. 2C and D). The displayed impedance curves in
Fig. 2C clearly confirmed the covalent attachment of NH2 termi-
nated sequence on the carboxylated MWCNT-COOH interface. As
seen, upon immobilization of the aptamer, the Rct value largely
increased (Fig. 2C, curve b) compared to that obtained the from
Nyquist plot for the non-bioconjugated composite interface
1502 M. Shamsipur et al. / Electrochimica Acta 283 (2018) 1498e1506
Fig. 2. Cyclic voltammograms (A) and Nyquist plots (B) recorded in the solution 0.1 M KCl 0.5 mM [Fe(CN)6]3/4- for GCE (a), GCE/nano fiber (b), GCE/NF/AuNPs (c) and GCE/NF/
AuNPs/MWCNTs (d). Cyclic voltammograms (C) and Nyquist diagrams (D) recorded at GCE/NF/AuNPs/WMCNTs (a), GCE/NF/AuNPs/MWCNTs/ssDNA (b) and GCE/NF/AuNPs/
MWCNTs/ssDNA after treatment with 1  103 cells mL1 of K562 cells (c).
(Fig. 2C, curve a). This signal enhancement was ascribed to the
physical coverage by the oligonucleotides and the resulting repul-
sive electrostatic-interaction between negatively charged phos-
phate backbones of the ssDNA with [Fe(CN)6]3/4-. The next FIS
measurement was accomplished at the K562 treated-biosensor,
where the prepared biosensor was soaked for the optimized incu-
bation time of 40 min in a solution of 1  103 cells mL1 of
K562 cells. In this case, the Rct value distinctly increased (Fig. 2C,
curve c), signifying the creation of an effective barrier due to
binding of the bulky targets to the recognition elements immobi-
lized on the platform, which suppresses the access of redox probe
to the surface of electrode. The corresponding CV results were
found to be in agreement with the obtained FIS spectra in the redox
probe solution (Fig. 2D).
To evaluate the role of PES and AuNPs on the performance of
cytosensor, we compared the corresponding impedance data
recorded at the GCE/PES-NF/AuNPs/MWCNT (Fig. 3A), GCE/PES-NF/
MWCNT (Fig. 3B) and GCE/AuNPs/MWCNT (Fig. 3C) toward
1  103 cells mL1 of K562 cells. The respective sensitivities were
found to be 1.06 U cell1, 0.487 U cell1, and 0.202 U cell1,
respectively, indicating the superior performance of GCE/PES-NF/
AuNPs/MWCNT in transduction of the occurred affinity events be-
tween the immobilized aptamer and K562 cells. The corresponding
calculated effective surface area found to decrease in the order of
GCE/PES-AuNPs > GCE/PES > GCE, which is consisting with the re-
sults of the biosensors prepared based on these platforms.
3.2. Optimization studies
In order to immobilize the K562-aptamer on the GCE/NF-
AuNPs/MWCNTs, primarily, the solutions containing different
concentrations of the aptamer were considered and the corre-
sponding binding events were monitored by FIS (see Figs. S1A and
B). The biosensor prepared with the immobilized concentration of
10 mM aptamer delivered approximately 95% of the highest
response, while, 40% increase in aptamer concentration (i.e., up to
14 mM) caused only 5% improvement in the response. Apart from
this, during frequent experimental tests, it was found that the
sensor constructed by 10 mM of aptamer revealed initial responses,
which were much more repeatable and more confident than those
of other biosensors fabricated using the higher concentration of
aptamer, most possibly because the increased probability of the
nonspecific interactions toward the surface. It caused the aggre-
gating of recognition elements (aptamers) so that, during the
measurements, will lead to some uncertainties and fluctuations in
the response.
Moreover, the effect of incubation time, as an imperative
parameter for the complex formation between recognition element
and target, was investigated for the different incubation times of
20, 30, 40, 50, 60 and 70 min. The corresponding impedance signal
drastically increased up to 40 min, while the longer incubation
times did not improve the DRct response significantly, so 40 min
was selected as the optimal incubation time during all measure-
ments (Figs. S1C and D).
3.3. Analytical performance of cytosensor
To evaluate the effect of different concentration of K562 cells on
the response of the biosensor, the respective Nyquist plots were
measured, after incubation (for 40 min) of aptamer in a buffer so-
lution of pH 7.4 containing different amounts of cells, over the
frequency range of 105e0.1 Hz, with AC amplitude of 5 mV and in a
 M. Shamsipur et al. / Electrochimica Acta 283 (2018) 1498e1506 1503

Fig. 3. Nyquist plots recorded in the solution 0.1 M KCl 0.5 mM [Fe(CN)6]3/4- for GCE/PES-NF/AuNPs/MWCNT (A), GCE/PES-NF/MWCNT (B) and GCE/AuNPs/MWCNT (C) toward
1  103 cells mL1 of K562 cells.

solution of 0.5 mM of the redox probe. A complete calibration curve [16,32e34]. Although more elucidation of such behavior aimed to
was obtained from the resulting impedance data for the proposed be studied by our research group.
biosensor over a concentration range of 1  102e7  107 cells mL1
(see Fig. 4A). In details, upon the successive increase in the cell
3.4. Single frequency measurements: shortening the measurement
population in the incubation solution, a new equilibrium was
time
established at the interfacial electrode/electrolyte region, where
the higher target concentrations induced the successful formation
In recent decades, Faradaic impedance measurements have
of an affinity complex between recognition element and the target
been extensively employed in monitoring charge transfer changes
cell. The event occurred impeded the interfacial electron transfer
caused by interfacial binding events at surface of improved trans-
process of [Fe(CN)6]3/4- and caused a gradual increase in the
ducers for bio-/sensing of various diverse analytes. Most of all
diameter of Nyquist plot's semicircle. Accordingly, the measured Rct
Nyquist plots that were assessed during these measurements
values were sketched against log K562 cell concentration (cell
encompass a semicircle portion that was followed by a diagonal
mL1) (Fig. 4B), representing an appropriate linear relationship
straight line (Warburg line) corresponding to the impedance of
over the concentration range from 1  102 to 7  107 cells mL1
diffusion electroactive species from bulk solution to the interfacial
with a correlation coefficient of 0.9941. The detection limit (DL) was
region. Warburg line typically emerges in low frequencies, where
calculated based on three times standard deviation of the blank
the lower rate of polarities changes allow conducting Faradaic
signals (3s) as 60 cells mL1.
process, leading to diffusion of electroactive spices, i.e., charged
These results signified the proper affinity of the aptamer to
redox probe, to the sensing layer confined at the surface of elec-
target at the transducer surface, which found to be improved or
trode. These events could induce gross non-specific signal changes
comparable to those of previously reported electrochemical assay
and limit the accuracy of quantitative sensing methods.
methods for K562 cancer cell (Table 1). From inset of Fig. 4B, it can
Using of mostly positive DC (>0.1 V vs. Ag/AgCl) potential in
infer that the interaction of such biochemical reactions as antibody-
working with negative charged probe Fe(CN)3/46 results in rear-
antigens, DNA hybridization, protein-protein interaction and
rangement of the negatively charged species in biosensing layer
protein-DNA interactions are predominates by MichaeliseMenten
and variation of charge transfer resistance, the changes that is
relations. This mechanism explains the binding events in
referred as “measurement-related Rct changes” [15]. Herein, during
biochemical systems, in which the binding process become slow
impedimetric bioassay procedure, we used lower concentration for
with increasing target concentration. In brief, the existence of such
redox probe (0.5 mM) and aborted Nyquist plot upon approaching
behavior can be regarded as the main reason that confines the
Warburg region to avoid any “measurement-related Rct changes”.
magnitude of signal change obtained for a fixed incubations time
We concluded that a shortening in the measurement time over a
1504 M. Shamsipur et al. / Electrochimica Acta 283 (2018) 1498e1506

recording one complete spectrum that could dramatically diminish

the measurement time from around several min to the times lower
than 10 s. In this work, the variation of total impedance (jZj) as a
function of K562 concentration at several frequencies, in low fre-
quencies region were explored and the obtained relations were
plotted in Fig. 5A. The relative variations in the slopes of jZj vs. log
[K562] were compared with the slope obtained by the calibration
from multi-sine measurements (Fig. 5B). It was shown that, in
different applied frequencies, the relative variation of the slopes is
less than 15%. Therefore, a calibration curve with a comparable
sensitivity can be constructed by recording only one jZj value for
each concentration. Utilizing this alternative strategy lead to a
considerable savings in time and easily working.

3.5. Reproducibility, repeatability, stability and specificity

Reproducibility of the evaluated signal was studied at the sur-

Fig. 4. (A) EIS responses of GCE/NF/AuNPs/MWCNTs/ssDNA after incubation with
different concentrations of K562 cancer cells. (B) The plot of Rct vs. log [K562] over the
concentration range of 1  102e7  107 cells mL1. Inset shows the signal relate to with
[K562].
face of GCE/NF/AuNPs/MWCNTs/ssDNA. The EIS response of the six
electrode constructed precisely through same procedure was
investigated by Faradaic impedance measurements. The relative
standard deviation (RSD) of the six measurement for particular
concentration of K562 cancer cells was calculated to be 2.87%,
indicating high reproducibility of the extended sensing device
under the optimized experimental condition. The repeatability of
the response was also considered for the biosensor after six mea-
surement for K562 treated electrode (i.e., 1000 cell mL1). The
relative standard deviation (RSD) was obtained as 2.16%. The sta-
bility of the constructed biosensor was finally investigated. After 18
day storing in dark (4  C), the signal change was evaluated and
signified that 89.7% of its initial response was retained, compared
with the freshly prepared electrode. The binding specificity be-
tween the fabricated biosensor and some possible interferences
including MCF-7, prostate-specific antigen (PSA), and A239 was
compared to the K562 response (Fig. 6). The corresponding changes
occurred in the transduced signal are shown on top of each bar as
percent. The increase in Rct for the bound complex of K562 and its
specific aptamer is quite obvious, while the change in charge
transfer resistance is minimal for typical selected interferences.

3.6. Human serum samples analysis

low range of frequency, just enough to attain the semicircle portion,
will be beneficial.
Particularly, in electrochemical driven protein-biosensing de-
vices, considering the fact that the population of massive target
proteins at the surface is variable in subsequent measurements, it is
difficult to monitor and recognize these nonspecific signal changes.
Therefore, exploiting a transduction approach that permit lower
occurrence of such non-specific signal changes can greatly im-
proves the accuracy of response. During this work, we also used a
single frequency measurement (SFM) by the recording the total
impedance (jZj) in an individual frequency [16,35,36], instead of
The application of the proposed biosensor in clinical laboratory
diagnosis was tested. The conventional sample preparations pro-
cedures was applied to prepare the serum sample for the analytical
investigation of the biosensor toward K562. In details, 3 mL
methanol was added to 2.5 mL of serum sample and, after vortexing
for 6 min, the precipitated proteins were separated by centrifuga-
tion for 3 min at 5500 rpm. The clear supernatant layer was filtered
through 0.45 mm milli-pore filter to obtain protein-free human
serum sample and its volume was adjusted to 12.5 mL using double
distilled water. Afterward, K562 cancer cells were spiked directly
into blood sample preparation and the standard addition method

Table 1
Merits of some comparable published biosensors developed for the determination of K562 cells.

Transduction signal LOD (cell mL1) Liner range (cell mL1) References

Electrochemical 1000 5  103e5  107 [37]

Electrochemical 3000 1  104e1  107 [38]
Electrochemical 500 7.5  102e3.9  106 [39]
Electrochemiluminescence 46 1  102e2  107 [40]
Electrochemical 79 1  102e1  107 [41]
Electrochemical 73 1  102e1  107 [42]
Electrochemical 30 1  102e1  107 [43]
Electrochemical 60 1  102e1  107 This work
 M. Shamsipur et al. / Electrochimica Acta 283 (2018) 1498e1506
Fig. 5. A) Variation of total impedance (jZj) as a function of [K562]. In several fre-
quency. B) The relative variation of the slopes of in either of individual frequencies in
comparison to the multi-sine calibration showed in Fig. 3.
Fig. 6. Comparison of the EIS responses of GCE/NF/AuNPs/MWCNTs/ssDNA by
employing different cells. (MCF-7, prostate-specific antigen (PSA), A239 and K562 cells
(1  104 cells mL1).
Table 2
Determination K562 cancer cells in blood sample.
Number of sample Spiking K562 cell (cells mL1)
1 3  103
2 1  104
3 2  105
4 2  106
1505
Fig. 7. The Nyquist plots of cytosensor in the solution 0.1 M KCl 0.5 mM [Fe(CN)6]3/4-
recorded without cell incubation (a) after incubating in healthy serum (b) and CML
serum patient (c).
was applied to measure the K562 cancer cells content using the EIS
method. The recovery of the K562 cancer cells was found to be
between 90.02 and 104.1% (Table 2), representing the appropriate
analytical performance of the method toward blood samples con-
taining K562.
Similar measurement were also conducted in serum sample of
CML patient and its response was compared with a normal sample
of a healthy person, a brief examination that disclosed the capa-
bility of the biosensors in identification of a CML patient. As shown
in Fig. 7, the Rct response of the biosensor before and after treat-
ment to the CML sample was drastically changed from 571 to
1217 U, while a similar procedure for the healthy sample shows that
the Nyquist plot did not show significant change in Rct (613 U).
4. Conclusions
We know that fast and quantitative detection of cancer cells can
take away a part of the cancer-related suffering from the patient's
side. In this study, a non-complicated and easy produced composite
was employed to support a single strand DNA able to monitor K562
cancer cells. We believes the composing of PES, AuNPs and
MWCNTs cannot introduce a new approach in material engineer-
ing, but the displayed performance of composite in conjugation
with the recognition element would promise us to reports a
powerful biosensing device for K562 in routine analyses. Although,
the procedure of signal transduction by recording the conventional
Nyquist is associated with some possible source of uncertainties
through signal recording, as is expressed in the discussion section,
we aimed challenges to counteract them by assisting SFM. This
procedure significantly saved time of transduction of the binding
events occurred at the electrode/electrolyte interface. The results
showed a promising a correlation in figure of merit when using
SFM in compared to its traditional multi-sine procedure.
Detected K562 cell (cells mL1) RSD% (n ¼ 3) (%) Recovery (%)
3.12  103 2.11 104.1
9.64  103 2.02 96.39
1.80  105 1.91 90.02
1.95  106 1.45 97.65
1506 M. Shamsipur et al. / Electrochimica Acta 283 (2018) 1498e1506
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.electacta.2018.07.089.
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