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1. Contents lists available at ScienceDirect
Microchemical Journal
journal homepage: www.elsevier.com/locate/microc
PAMAM dendrimer modified screen printed electrodes for impedimetric
detection of miRNA-34a
Gulsah Congur, Arzum Erdem
⁎
Faculty of Pharmacy, Analytical Chemistry Department, Ege University, 35100 Bornova, Izmir, Turkey
The Institute of Natural and Applied Sciences, Biotechnology Department, Ege University, 35100 Bornova, Izmir, Turkey
A R T I C L E I N F O
Keywords:
PAMAM dendrimer
Screen printed electrode (SPE)
miRNA-34a
Electrochemical impedance spectroscopy (EIS)
A B S T R A C T
In this study, the effective usage of PAMAM dendrimer was implemented for detection of microRNA-34a
(miRNA-34a) which was known as a biomarker for many vital diseases such as cancer, Alzheimer, etc. The
microscopic and electrochemical characterization of the PAMAM modified electrode surface was performed
successively. The experimental conditions such as DNA probe, target concentration, hybridization time were
optimized for the purpose of effective nucleic acid hybridization between miRNA-34a RNA target and its
complementary DNA probe. Under optimum conditions, the selectivity of the impedimetric nucleic acid bio-
sensor was tested in the presence of microRNA-15a (miRNA-15a) and microRNA-660 (miRNA-660). Our im-
pedimetric biosensor platform could be selectively detect its target, miRNA-34a even if the hybridization was
done in fetal bovine serum (FBS), which served a complex media for detection process. This disposable, easy-to-
use, cheap and time saving impedimetric biosensor platform is the first study in the literature on behalf of
electrochemical detection of miRNA-34a.
1. Introduction
Dendrimers are constituted of a central core-shell and decorated
with branched units (terminal groups) which are varied and repeated
several times that makes them reach in various size. Several types of
(bio)molecules can interact and bind with dendrimers or dendrimer
modified surfaces due to their branched units (terminal groups) which
effect physical and chemical properties of dendrimers [1]. Dendrimer
modification ensures enhanced surface area by terminal groups.
Therefore, the modification of dendrimer at biosensor surface allows to
make detection of target molecules such as proteins, drugs or nucleic
acids sensitively and selectively by reaching low detection levels [2–9].
At this point, electrochemical techniques are able to detect their targets
sensitively, selectively and in a reliable way with a fast sensor response
compared to conventional techniques [9].
The first recognition of microRNAs (miRNAs) was performed in
Caenorhabditis elegans and miRNAs were defined as “a class of short
endogenous non-coding RNAs”. They are single stranded RNAs having
18–25 bases. They are also known as non-coding nucleic acids, but they
are involved in crucial roles in cells such as proliferation and apoptosis.
Today, several vital diseases including cancer, neurodegenerative dis-
eases, diabetes and heart diseases are linked with the up/down-reg-
ulation of miRNAs in related pathways [10,11]. Thus, the monitoring of
miRNAs has gained great importance day-by-day and there has been
numerous reports about detection of miRNAs using biosensors [12–18].
Guo et al. [18] reported bifunctional nanostructures using gold nano-
particle (GNP) coupled amine-terminated dendrimer modified gold chip
for detection of miRNA via frequency response analysis. Liu et al. [15]
reported an electrochemical biosensor based on chitosan-graphene
composite, gold and silver nanocluster and generation 3.5 poly(ami-
doamine) (PAMAM) dendrimer modified glassy carbon electrode to
detect miRNA-126. The interaction of digoxin and antidigoxin antibody
labeled with horse radish peroxidase was detected by differential pulse
voltammetry (DPV) technique. Catalytic hairpin assembly constructed
gold electrode was used to monitor miRNA [12]. The enzymatic reac-
tion of streptavidin-alkaline phosphatase and α-naphthyl phosphate
occurred after the hybridization of hairpin probe and target miRNA was
detected using electrochemical impedance spectroscopy (EIS) and
square wave voltammetry (SWV) techniques.
EIS has received an increasing attraction for its implementation to
biosensors. The interfacial changes occurred between analyte and bio-
sensor surface can be traced using this technique [19]. Each mod-
ification/immobilization step onto the surface of the electrode used as a
biosensor surface make the resistance changes depending upon the
electron transfer between the electrode and analysis solution. Since it is
a powerful detection technique for the monitoring of biomolecular
https://doi.org/10.1016/j.microc.2019.05.040
Received 17 December 2018; Received in revised form 16 April 2019; Accepted 15 May 2019
⁎
Corresponding author at: Faculty of Pharmacy, Analytical Chemistry Department, Ege University, 35100 Bornova, Izmir, Turkey.
E-mail address: arzum.erdem@ege.edu.tr (A. Erdem).
Microchemical Journal 148 (2019) 748–758
Available online 16 May 2019
0026-265X/ © 2019 Elsevier B.V. All rights reserved.
T

2. interactions such as drug-nucleic acid interactions, protein-antibody or
aptamer interactions, and nucleic acid hybridizations, there are many
reports in the literature about development of impedimetric biosensors
[2,4–7,20–23].
Herein, the impedimetric biosensor was reported to detect miRNA-
34a in a selective and sensitive way using poly(amidoamine) PAMAM
dendrimer modified single-use screen printed carbon electrode (SPE).
miRNA-34a is a biomarker for vital diseases such as Alzheimer [24] and
various types of cancer [25–27]. Amino linked DNA probe specifically
designed for miRNA-34a was immobilized at the PAMAM dendrimer
modified SPE surface, then, the hybridization of DNA probe and its
miRNA target occurred at the electrode surface. Each immobilization/
modification step was monitored by electrochemical impedance spec-
troscopy (EIS) technique. The selectivity of the impedimetric biosensor
was tested in the presence of miRNA-15a and miRNA-660. Our bio-
sensor was sensitively and selectively detected its target, miRNA-34a,
even if the hybridization was performed in artificial serum, fetal bovine
serum (FBS).
2. Experimental
The experimental scheme presenting the modification of SPE sur-
face by G2-PS and the hybridization occurred at the surface of G2-PS/
SPE was given in Scheme 1.
2.1. Apparatus
AUTOLAB–PGSTAT 302 supplied with a FRA 2.0 module and GPES
4.9 software package (Eco Chemie, The Netherlands) was used as an
electrochemical analysis system to carry out all voltammetric and im-
pedimetric measurements which were performed in the Faraday cage
(Eco Chemie, The Netherlands).
The dimension of planar screen-printed electrode (SPE) was
3.4 cm × 1.0 cm × 0.05 cm (length × width × height). It consisted of
three main parts; a graphite counter electrode, a graphite working
electrode (4 mm in diameter), and a silver pseudo reference electrode.
These single-use SPEs were commercially purchased from DropSens
(Oviedo-Asturias, Spain). The connection between SPEs and the po-
tentiostat could be achieved by a specific DropSens connector (ref.
DSC). A 35 μL droplet covered the working area was placed for mea-
surements onto SPEs.
2.2. Chemicals
The amino-linked single stranded miRNA-34a DNA probe and its
complementary miRNA-34a target, or miRNA-15a and miRNA-660
which were used for testing the selectivity were purchased from TIB-
MOLBIOL (Berlin, Germany). The second generation poly(amidoamine)
(PAMAM) dendrimer with 1,4-diaminobutane core (G2-PS) which has
16 succinamic acid surface groups, fetal bovine serum (FBS) as the
artificial serum, potassiumhexacyanoferrate(III) (K3Fe(CN)6) and po-
tassiumhexacyanoferrate(II) trihydrate (K4Fe(CN)6 · 3H2O) were pur-
chased from Sigma. All oligonucleotide (ODN) stock solutions were
prepared as 500 μg/mL in Tris-EDTA buffer solution (10 mM Tris–HCl,
1 mM EDTA, pH 8.00; TE) and kept frozen for stability of them. More
diluted solutions of ODNs were prepared with 50 mM phosphate buffer
solution (PBS; pH 7.40) according to the hybridization protocol. For the
preparation of these buffer solutions, ultrapure water (as RNase/DNase
free) was used. Analytical reagent grade chemicals were used supplied
from Sigma and Merck.
Amino linked miRNA-34a DNA probe (22 bases):
5′–NH2-ACA ACC AGC TAA GAC ACT GCC A-3′
miRNA-34a RNA target (22 bases):
5′–UGG CAG UGU CUU AGC UGG UUG U-3′
miRNA-15a RNA sequence (22 bases):
5′–UAG CAG CAC AUA AUG GUU UGU G-3′
miRNA-660 RNA sequence (22 bases):
5′–UAC CCA UUG CAU AUC GGA GGU G-3′
2.3. Scanning electron microscopy (SEM) analysis for microscopic
characterization of electrode surface
SEM analysis of SPEs was performed by field emission scanning
electron microscope (FE-SEM) (Quanta 400FEI, Tokyo, Japan).
Scheme 1. Schematic representation of the development of G2-PS modified single-use SPEs and the impedimetric detection of miRNA-34a RNA target using G2-PS/
SPEs.
G. Congur and A. Erdem Microchemical Journal 148 (2019) 748–758
749

3. Required acceleration voltage was 5.00 kV and the resolution varied
from 1 μm to 10 μm.
2.4. G2-PS preparation
G2-PS solution was prepared and used for the modification of SPE
surface as described and optimized in our previous study [4]. The
frozen stock solution of G2-PS (106
μg/mL) was diluted in PBS (pH 7.4)
as 1 μg/mL.
2.5. Preparation of SPEs
2.5.1. The modification of G2-PS onto SPE surface
The 35 μL droplet of 1 μg/mL G2-PS covered to unpretreated SPE
surface during 1 h modification time. The unbound G2-PS molecules
were removed by rinsing each of the electrodes with PBS (pH 7.40) for
5 s [4].
2.5.2. miRNA-34a DNA probe immobilization on G2-PS/SPE surface
35 μL of the required amount of amino linked miRNA-34a specific
DNA probe solution covered to the surface of G2-PS/SPE during 30 min.
The covalent coupling between carboxylated ends of the G2-PS and
amine group of DNA probe could be achieved [4,28] which was re-
sulted in the immobilization of DNA probe at the surface of G2-PS/SPE.
The elimination of the unspecific binding was achieved by washing the
electrodes with PBS (pH 7.40).
2.5.3. Hybridization between amino linked miRNA-34a DNA probe and its
complementary miRNA target or noncomplementary miRNAs (miRNA-15a/
miRNA-660)
35 μL droplet of required amount of miRNA-34a, or miRNA-15a, or
miRNA-660 covered at the surface of DNA probe immobilized G2-PS/
SPEs during 30 min. The electrodes were then washed with PBS
(pH 7.40) to prevent unspecific hybridization.
2.6. Voltammetric measurements
The cyclic voltammetry (CV) measurements were performed by
scanning from −0.60 to +1.00 V and the scan rate as 50 mV/s. All
measurements were done in 35 μL of 2 mM K3[Fe(CN)6]/K4[Fe(CN)6]
(1:1) containing 0.1 M KCl.
2.7. Impedimetric measurements
2.50 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture in 0.10 M KCl was
prepared as a redox probe and 35 μL of this probe was used for all
impedimetric measurements. The impedance was measured in the fre-
quency range from 100 mHz to 100 kHz at a potential of +0.23 V with
a sinusoidal signal of 10 mV. The frequency interval was divided into 98
logarithmically equidistant measure points. The respective semicircle
diameter corresponds to the charge-transfer resistance, Rct, the values
of which are calculated using the fitting programme AUTOLAB 302
(FRA, version 4.9 Eco Chemie, The Netherlands). All measurements
were performed in the Faraday cage (Eco Chemie, The Netherlands).
Fig. 1. SEM images of unmodified (A), 1.00 μg/mL G2-PS modified SPE (B) by using the identical acceleration voltage as 5.00 kV with resolution 10 μm (a), 5 μm (b)
and 1 μm (c).
G. Congur and A. Erdem Microchemical Journal 148 (2019) 748–758
750

4. Fig. 2. Nyquist diagrams (A), representing the charge
transfer resistance (Rct) values and histograms (B) re-
presenting the average Rct values (n = 3) obtained by
unmodified SPE (a), 1.00 μg/mL G2-PS modified SPE (b),
2.00 μg/mL amino linked miRNA-34a DNA probe im-
mobilized G2-PS/SPE (c) by using electrochemical im-
pedance spectroscopy (EIS). Inset: The equivalent circuit
model used to fit the impedance data. Its parameters are
listed in the text.RS represents the solution resistance. The
constant phase element, Q, is related to the space charge
capacitance occurred at the electrode–electrolyte inter-
face. Rct is the charge transfer resistance occurred at the
electrode–electrolyte interface. The constant phase ele-
ment, W, is the Warburg impedance due to mass transfer
to the electrode surface.
Fig. 3. Nyquist diagrams obtained by EIS technique,
and represents the nucleic acid hybridization of 0.05
(A); 0.10 (B); 0.25 (C); 0.50 μg/mL (D) amino linked
miRNA-34a DNA probe and 10 μg/mL miRNA-34a
target at the surface of G2-PS/SPE. SPE (a), G2-PS/
SPE (b), DNA probe immobilized G2-PS/SPE (c),
nucleic acid hybridization between DNA probe and
miRNA-34a target at the surface of G2-PS/SPE (d).
Inset was the equivalent circuit model used to fit the
impedance data.
G. Congur and A. Erdem Microchemical Journal 148 (2019) 748–758
751

5. 3. Results and discussion
The SPE surface before/after G2-PS modification (Fig. 1-A and B,
respectively) was firstly characterized by a microscopic technique,
SEM. The surface coverage of SPE with G2-PS could be clearly seen at
different resolutions. The surface roughness of unmodified SPE was
monitored in Fig. 1-A. The surface of SPE became more smooth and
bright [4] due to coverage of G2-PS which could be seen in Fig. 1-A, b
to B, b. However, this coverage can have been achieved in a partial
manner due to wet adsorption process (Fig. 1-A, c to B, c).
After the microscopic characterization step, the electrochemical
characterization was performed by EIS (Fig. 2) and CV (Fig. S1) tech-
niques before/after G2-PS modification of the SPEs. The charge transfer
resistance (Rct) was measured by EIS technique and the average Rct was
found to be 1128.50 ± 248.60 Ohm (RSD% = 22.00%, n = 21) by
unmodified SPEs (Fig. 2-B, a). 0.62 fold higher average Rct value could
be obtained (Fig. 2-B, b) and measured as 1839.20 ± 347.00 Ohm
(RSD% = 18.90%, n = 18) by successful modification of 1 μg/mL G2-
PS at SPE surface. The modification of G2-PS at the surface of SPE was
proved by the change at the Rct obtained after G2-PS modification onto
the SPE surface, which was in parallel with the SEM results (Fig. 1).
This increase at Rct value could be attributed to negatively charged
carboxylated ends of G2-PS introduced during passive adsorption pro-
cess at the surface of SPEs [4,28]. The repulsive electrostatic interaction
between these negatively charged ends of G2-PS and ferricyanide ions
ended up the increase at the Rct [4,28–30]. After the immobilization of
2 μg/mL amino linked miRNA-34a DNA probe at the surface of G2-PS/
SPE, 2.65 fold higher average Rct value was obtained (Fig. 2-B, c) and
measured as 6874.00 ± 816.00 Ohm with the RSD% as 11.90%
(n = 3). This increase at the Rct value was a proof of the interaction
between negatively charged backbone of DNA probe and negatively
charged ferricyanide ions [4,6].
The apparent fractional coverage (QR
IS
) of G2-PS modified SPE and
2 μg/mL amino linked miRNA-34a DNA probe immobilized G2-PS/SPE
was calculated using Eq. (1) [31] and found to be 0.30 and 0.75, re-
spectively (Table S1). These results indicated the surface coverage of
G2-PS and DNA probe.
=
Q 1
R
R
IS
R ct
SPE
ct
G2 PS/SPE
(1)
CV measurements were performed before/after G2-PS modification
of SPEs and also performed before and after immobilization of 2 μg/mL
amino linked miRNA-34a DNA probe at the surface of unmodified and
G2-PS modified SPEs (Fig. S1). The anodic peak currents (Ia) and cal-
culated surface areas (A) were given in Table S2. The average Ia was
obtained by unmodified SPEs as 32.50 ± 1.90 μA (RSD% = 6.00%,
n = 3) (Fig. S1-B, a). After the immobilization of miRNA-34a DNA
probe at the surface of unmodified SPE, the average Ia was lower as
14.0% and measured as 27.70 ± 1.10 μA (RSD% = 4.00%, n = 3) (Fig.
S1-B, b). This decrease at Ia may be attributed the repulsive interaction
between negatively charged backbone of single-stranded DNA probe
and ferricyanide ions [7,33,34,36]. After the modification of G2-PS at
the surface of SPE, the average Ia was measured as 31.80 ± 0.20 μA
with the RSD% = 0.70% (n = 3) (Fig. S1-B, c). After the immobiliza-
tion of miRNA-34a DNA probe at the surface of G2-PS/SPE, the average
Ia was lower as 16.00% and measured as 26.60 ± 1.10 μA (RSD
% = 2.00%, n = 3) (Fig. S1-B, d). These results were strong proof for
the modification G2-PS at the surface of SPE, and the immobilization of
DNA probe unmodified and G2-PS modified SPE. However, the im-
mobilization of DNA probe at G2-PS/SPE surface could be more effec-
tive according to the Ia with better reproducibility as 2.00% and de-
crease ratio of the Ia.
Next, the concentration of amino-linked miRNA-34a DNA probe was
optimized using EIS technique (Fig. 3). The real (Z′) and imaginary
Fig. 4. Nyquist diagrams (A) and histograms (B) obtained
by SPE (a) G2-PS/SPE (b), 0.10 μg/mL amino linked
miRNA-34a DNA probe immobilized G2-PS/SPE (c), after
the nucleic acid hybridization between 0.10 μg/mL amino
linked miRNA-34a DNA probe and 10 μg/mL miRNA-34a
target at the surface of G2-PS/SPE using 15 (d), 30 (e)
and 60 min (f) hybridization times. Inset was the
equivalent circuit model used to fit the impedance
data.Impedimetric analysis were performed by three re-
petitive measurements (n = 3).
G. Congur and A. Erdem Microchemical Journal 148 (2019) 748–758
752

6. components (Z″) consist of the complex impedance. [35]. The impedi-
metric results were fitted by using an equivalent circuit model (i.e.,
inset in each figure). Rct is the charge transfer resistance defined as the
resistance which depends on the dielectric and insulating character-
istics at the electrode/electrolyte interface. The electron transfer was
limited at higher frequencies. The linear section could be seen at lower
frequencies which may be attributed to the diffusion [35]. The hy-
bridization of miRNA-34a DNA probe at different concentrations from
0.05 to 0.50 μg/mL and 10 μg/mL miRNA-34a target was performed
during 30 min at the surface of G2-PS/SPE. The impedimetric mea-
surements were done after each immobilization/hybridization step. The
change ratios of the Rct after immobilization of miRNA-34a DNA probe
at different concentration levels and after the hybridization step were
given in Table S3. The Rct was measured as 1635, 2875, 4314, 4250
Ohm after the immobilization of 0.05, 0.10, 0.25 and 0.50 μg/mL
amino-linked miRNA-34a DNA probe, respectively. After the hy-
bridization of 0.05, 0.10, 0.25 and 0.50 μg/mL amino-linked miRNA-
34a DNA probe and 10 μg/mL miRNA-34a target, the Rct was obtained
as 4111, 4567, 5299 and 3886 Ohm, respectively. The 0.78 fold in-
crease at the Rct value was obtained after the immobilization of
0.10 μg/mL DNA probe and 0.49 fold increase at the Rct value was
obtained after the hybridization of 0.10 μg/mL DNA probe and 10 μg/
mL miRNA-34a target (Table S3). The highest increase at the Rct both
the immobilization of DNA probe and the hybridization of DNA probe
and miRNA target could be obtained in the presence of 0.10 μg/mL
miRNA-34a DNA probe (Fig. 3-B, c to d). The average Rct values after
the immobilization of 0.10 μg/mL DNA probe and after the hybridiza-
tion at this concentration level of DNA probe were obtained as
2702.30 ± 331.60 Ohm (RSD% = 12.30%, n = 3) and
4315.00 ± 290.70 Ohm (RSD% = 6.70%, n = 3), respectively (Fig.
S2). 0.10 μg/mL miRNA-34a DNA probe concentration was chosen as
optimum. Further studies were utilized at this DNA probe concentra-
tion.
The optimization of hybridization time was performed in the next step
(Fig. 4). The hybridization of 0.10 μg/mL amino linked miRNA-34a DNA
probe and 10 μg/mL miRNA-34a target was performed during 15, 30 and
60 min hybridization times. The highest Rct value was obtained after
30 min hybridization (Fig. 4-A, e) and the average Rct value was found to
be 4302.80 ± 596.20 Ohm (RSD% = 13.90%, n = 3) (Fig. 4-B, e). The
hybridization time was optimized as 30 min in this experimental step.
After the optimization of DNA probe and hybridization time, the
most effective nucleic acid hybridization condition was finally achieved
by optimization of the miRNA-34a target concentration (Fig. 5). For this
purpose, 0.10 μg/mL miRNA-34a DNA probe and miRNA-34a target at
Fig. 5. Nyquist diagrams obtained by (A) SPE (a), G2-PS/
SPE (b), 0.10 μg/mL miRNA-34a DNA probe immobilized
G2-PS/SPE (c), after hybridization of 0.10 μg/mL miRNA-
34a DNA probe and 2.50 (d); 5.00 (e); 7.50 (f); 10 μg/mL
(g) miRNA-34a target at the surface of G2-PS/SPE. Inset
was the equivalent circuit model used to fit the im-
pedance data. Line graph (B) representing the average Rct
values (n = 3) obtained after hybridization of 0.10 μg/mL
miRNA-34a DNA probe and miRNA-34a target at different
concentration levels. Inset was the calibration graph
based on the Rct values obtained after the hybridization of
0.10 μg/mL amino linked miRNA-34a DNA probe and
miRNA-34a target at different concentrations ranging
from 0 to 7.50 μg/mL at the surface of G2-PS/SPEs
(n = 3).
G. Congur and A. Erdem Microchemical Journal 148 (2019) 748–758
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7. different concentration levels from 0 to 10 μg/mL at the surface of G2-
PS/SPE was hybridized. Highest Rct value could be obtained after the
hybridization of 0.10 μg/mL miRNA-34a DNA probe and 7.50 μg/mL
miRNA-34a target (Fig. 5-A, c to f) and the average Rct was found to be
4623.50 ± 642.80 Ohm (RSD% = 13.90%, n = 3) (Fig. 5-B). Thus, the
optimum concentration level of miRNA-34a target was chosen as
7.50 μg/mL.
A linear graph was obtained in the concentration range between 0
and 7.5 μg/mL (shown in Fig. 5B, inset). The detection limit (DL) was
calculated according to the method described by Miller and Miller [36],
and found to be 0.95 μg/mL (equals to 135 nM) with the coefficient of
determination (R2
) = 0.9903 (Fig. 5B, inset).
The selectivity of the G2-PS/SPE based impedimetric biosensor was
then tested in the presence of miRNA-15a and miRNA-660 under
optimum conditions (Fig. 6). The ΔRct was calculated as in Eq. (2).
=
R R R
ct ct2 ct1 (2)
Rct2 represents the Rct value obtained after hybridization of miRNA-
34a DNA probe and miRNA target (Fig. 6I-A, B, C-d), Rct1 represents the
Rct value obtained after immobilization of miRNA-34a DNA probe at
the surface of G2-PS/SPE (Fig. 6I-A, B, C-c).
The average ΔRct values were represented in Fig. 6-B. After the
hybridization of miRNA-34a DNA probe and its RNA target or miRNA-
15a and miRNA-660, the average ΔRct values were estimated as
1479.80 ± 258.70 Ohm, 1147.50 ± 173.20 Ohm and
865.00 ± 63.60 Ohm, respectively. The highest average ΔRct value
was obtained in the presence of the hybridization between miRNA-34a
DNA probe and its miRNA target with the RSD% = 17.90% (n = 3). It
Fig. 6. Nyquist diagrams (I) obtained by nucleic acid hybridization between 0.10 μg/mL miRNA-34a DNA probe and 7.50 μg/mL miRNA-34a target (A), miRNA-15a
(B) and miRNA-660 (C) at the surface of G2-PS/SPE. SPE (a), G2-PS/SPE (b), miRNA-34a DNA probe immobilized G2-PS/SPE (c), the hybridization between DNA
probe and miRNA sequence (d). Inset was the equivalent circuit model used to fit the impedance data. Histograms (II) representing the average ΔRct values obtained
after the hybridization of 0.10 μg/mL miRNA-34a DNA probe and 7.5 μg/mL miRNA-34a (a), miRNA-16 (b), miRNA-660 (c) at the surface of G2-PS/SPE (n = 3).
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8. can be concluded that our impedimetric biosensor can sensitively and
selectively detect the target miRNA-34a sequence.
Serum is known as a complex biological fluid which contains var-
ious compositions such as hormones, electrolytes, nutrients and pro-
teins (albumins, globulins, etc.) [33]. The selective detection of target
molecule in such a complex media as serum was also under the aim of
this study. Therefore, fetal bovine serum (FBS) was used as a complex
media to detect miRNA-34a target by using developed impedimetric
biosensor. First, the dilution of FBS with PBS was studied in the pre-
sence of DNA-RNA hybridization (Fig. S3). For this purpose, the diluted
FBS solutions were prepared by PBS (pH 7.4) in different ratios of
FBS:PBS as1:10, 1:100, 1:1000. Then, hybridization of 0.10 μg/mL
miRNA-34a DNA probe and 7.50 μg/mL miRNA-34a RNA target was
performed in PBS (pH 7.4) (Fig. S3-d) or 1:10 (Fig. S3-e), 1:100 (Fig. S3-
f), 1:1000 (Fig. S3-g) diluted FBS:PBS solutions. The Rct was obtained as
4827 Ohm (Fig. S3-d) after the hybridization occurred in PBS at G2-PS/
SPE surface (pH 7.4). The Rct value measured after the hybridization
occurred in 1:10 FBS:PBS diluted solution was obtained as 5812 Ohm
(Fig. S3-e) which was the closest Rct value compared to the ones ob-
tained in 1:100 and 1:1000 diluted FBS:PBS solutions. Thus, further
studies were performed in 1:10 diluted FBS:PBS solution.
In the next step, the effect of miRNA-34a target concentration onto
hybridization process was studied in 1:10 diluted FBS:PBS solution
(Fig. 7). The hybridization of 0.1 μg/mL miRNA-34a DNA probe and
miRNA-34a target at different concentration levels from 0.5 to 3 μg/mL
was done in 1:10 diluted FBS:PBS solution at G2-PS/SPE surface. The
ΔRct values were calculated as in Eq. (2) and represented in a line graph
in Fig. 7. The highest ΔRct value could be obtained in the presence of
2.5 μg/mL miRNA-34a target concentration (Fig. 7A-h). The average
ΔRct value was estimated as 4749.0 ± 561.44 Ohm (RSD% = 11.8%,
n = 3) (Fig. 7B). Thus, 2.5 μg/mL miRNA-34a target concentration was
chosen as optimum for further studies.
The DL was also calculated in the concentration range from 1.00 to
2.50 μg/mL (Fig. 7B, inset) according to the equation described by
Miller and Miller [36], and found to be 0.52 μg/mL (equals to 74 nM).
The selectivity of our impedimetric biosensor in 1:10 diluted
FBS:PBS solution was then tested against miRNA-15a and miRNA-660
(Fig. 8). The hybridization was performed of 0.10 μg/mL miRNA-34a
DNA probe and 2.50 μg/mL its RNA target or other RNA sequences. The
ΔRct values were calculated as in Eq. (2). The ΔRct values were found to
be 4750.50 ± 563.60 Ohm, 3977.50 ± 243.90 Ohm and
4077.00 ± 414.30 Ohm after the hybridization of miRNA-34a DNA
probe and miRNA-34a target, miRNA-15a or miRNA-660, respectively.
It can be concluded that the impedimetric biosensor developed using
G2-PS/SPE was selectively detected its target, miRNA-34a, even in a
complex media, FBS.
Fig. 7. Nyquist diagrams (A) of SPE (a), G2-PS/SPE (b),
0.10 μg/mL miRNA-34a DNA probe immobilized G2-PS/
SPE (c), the nucleic acid hybridization between 0.10 μg/
mL miRNA-34a DNA probe and 0.50 (d); 1.00 (e); 1.50
(f); 2.00 (g); 2.50 (h), 3.00 (i) μg/mL miRNA-34a target
in 1:10 FBS:PBS (pH 7.4) at the surface of G2-PS/SPE.
Inset was the equivalent circuit model used to fit the
impedance data. Line graph (B) representing the average
ΔRct values (n = 3) obtained after the nucleic acid hy-
bridization between 0.1 μg/mL miRNA-34a DNA probe
and miRNA-34a target at different concentrations ranging
from 0.50 to 3.00 mg/mL in 1:10 FBS:PBS (pH 7.4) at the
surface of G2-PS/SPEs (n = 3). Inset was the calibration
graph based on the Rct values obtained after the hy-
bridization of 0.10 mg/mL amino linked miRNA-34a DNA
probe and miRNA-34a target at different concentrations
ranging from 1.00 to 2.50 mg/mL in 1:10 FBS:PBS
(pH 7.4) at the surface of G2-PS/SPEs (n = 3).
G. Congur and A. Erdem Microchemical Journal 148 (2019) 748–758
755

9. 4. Conclusion
Since the introduction of dendrimer molecules into biosensor
area, they have gained a great potential for development of elec-
trochemical biosensors due to they provide some crucial properties
such as good biocompatibility, having adequate functional groups to
generate chemical bonds and to obtain robust, large surface area
[2,15,29,34,37–39]. Herein, the second generation poly(amidoa-
mine) (PAMAM) dendrimer with 1,4-diaminobutane core (G2-PS)
was modified at the surface of single-use screen printed carbon
electrode surface (SPE) and this modified electrode was applied for
development of a DNA biosensor, which was capable of electro-
chemical detection of miRNA-34a. The detection system was based
on the nucleic acid hybridization occurred between DNA probe and
its RNA target. Based on this strategy, miRNA-34a could be specifi-
cally detected using electrochemical impedance spectroscopy (EIS)
technique in repetitive measurements, which provides accurate im-
pedance data before and after hybridization and is widely used for
monitoring of biointeraction process [4,8,15,39,40]. Moreover,
selective and sensitive detection of miRNA-34a could be achieved in
a complex media, FBS.
The reports in the literature for the purpose of detection of miRNAs
were given in Table 1. The most of the biosensors were developed for
miRNA-21 which was a model target for different types of cancers, such
as breast, liver, brain, prostate, myometrial cancers and also in cardio-
vascular diseases [12,41–46]. Gold electrode was used mostly for fabri-
cation of the biosensor developed for miRNA-21 detection
[12,41–44,46,47] which required polishing and washing steps with
strong oxidizing agents like Piranha solution. These steps were labor
intensive and time requiring; at least 12 h were needed for biosensor
preparation [43]. miRNA-203 was detected using quartz crystal micro-
balance (QCM) technique using gold chip [18]. DNA/gold nanoparticle/
dendrimer composite was synthesized and immobilized at gold surface.
Although a low detection limit (1 pM) could be obtained in that study,
the preparation of the biosensor required 50 h. Moreover, the usage of
extra chemical agents was needed for the preparation of gold chip. Be-
cause of the gold chip was not single-use, the design of the QCM based
biosensor could not be convenient for miniaturization. In another study,
Fig. 8. Nyquist diagrams (I) obtained by
nucleic acid hybridization between 0.10 μg/
mL miRNA-34a DNA probe and 2.50 μg/mL
miRNA-34a target (A), miRNA-15a (B) and
miRNA-660 (C) in 1:10 FBS:PBS (pH 7.4) at
the surface of G2-PS/SPE. SPE (a), G2-PS/
SPE (b), miRNA-34a DNA probe im-
mobilized G2-PS/SPE (c), the hybridization
between DNA probe and miRNA sequence
(d). Inset was the equivalent circuit model
used to fit the impedance data. Histograms
(II) representing the average ΔRct values
obtained after the hybridization of 0.10 μg/
mL miRNA-34a DNA probe and 2.50 μg/mL
miRNA-34a (a), miRNA-16 (b), miRNA-660
(c) RNA sequences in 1:10 FBS:PBS (pH 7.4)
at the surface of G2-PS/SPE (n = 3).
G. Congur and A. Erdem Microchemical Journal 148 (2019) 748–758
756

10. glassy carbon electrode (GCE) was used for voltammetric detection of
miRNA-126 [15]. GCE was sonicated, polished and then, the modifica-
tion of dendrimer, gold and silver nanoclusters, a chitosan-graphene
composite could be performed. Total preparation time of this biosensor
platform was 21 h requiring time-consuming experimental steps, i.e.,
PNA immobilization for 16 h. Moreover, this biosensor platform was not
appropriate for single-usage. It was clearly seen that the rest of the ap-
plications [12,15–18,39–50] reported in Table 1 were time-consuming,
labor-intensive and required the usage of extra chemical agents. In our
previous studies [17,40], pencil graphite electrode (PGE) was used for
detection of miRNA-34a. Longer preparation time (2 h 45 min and 4 h,
respectively) was needed for development of graphene oxide (GO)
modified PGEs. Also, the detection limits were higher than the one ob-
tained in this work. Mandli et al. [50] developed an impedimetric bio-
sensor platform for detection of miRNA-34a using polypyrrole (PPy)
modified PGEs. The DNA probe immobilization was performed in stirred
solution which required high volume of sample at higher concentration
level (5.0 μg/mL) compared to the one used in this study (0.1 μg/mL).
DNA probe immobilized PGEs were dipped into 100 μL miRNA-34a
contained samples. Compared to the study of Mandli et al. [50], less
sample required for development of G2-PS modified SPEs. The mod-
ification of G2-PS, the hybridization process and measurements required
approximately 2 h without necessity of using complex chemical agents
and labor-intensive process, which makes our disposable impedimetric
biosensor had a great potential for application of microchip technology
in the next step. At this point, our study has the advantage in comparison
to other reports [12,15–17,41–50].
In summary, a portable, disposable, easy-to-use, time saver and
cheap impedimetric biosensor was developed based on G2-PS/SPEs in
this study. To the best of our knowledge, this is the first study in the
literature for monitoring of miRNA-34a by using EIS technique. This
study is thought to lead to the further studies for monitoring of different
types of miRNAs or other biomolecules such as proteins, drugs and
nucleic acids.
Acknowledgments
A.E would like to express her gratitude to the Turkish Academy of
Sciences (TUBA) as a Principal member for its partial support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.microc.2019.05.040.
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