A review on the electrochemical biosensors for determination of microRNAs

Abstract

MicroRNAs (miRNAs) are a family of non-protein-coding, endogenous, small RNAs. They are a group of gene regulators that function mainly by binding the 3′ untranslated regions of specific target messenger RNA (mRNA) leading to gene inactivation by repression of mRNA transcription or induction of mRNA. Mature miRNAs are short molecules approximately 22 nucleotides in length. They regulate a wide range of biological functions from cell proliferation and death to cancer progression. Cellular miRNA expression levels can be used as biomarkers for the onset of disease states and in gene therapy for genetic disorders. Methods for detection of miRNA mainly include northern blotting, microarray, polymerase chain reaction (PCR). This review focuses on the use of electrochemical biosensors for the detection of microRNA.

Full text

Review
A review on the electrochemical biosensors for determination
of microRNAs
Ezat Hamidi-Asl
a,b,
n
, Ilaria Palchetti
b
, Ehteram Hasheminejad
a
, Marco Mascini
b
a
Eletroanalytical Chemistry Research Laboratory, Department of Analytical Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran
b
Università degli Studi di Firenze, Dipartimento di Chimica, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
article info
Article history:
Received 11 December 2012
Received in revised form
22 March 2013
Accepted 26 March 2013
Available online 17 April 2013
Keywords:
MicroRNAs
Electrochemistry
Biosensors
abstract
MicroRNAs (miRNAs) are a family of non-protein-coding, endogenous, small RNAs. They are a group of
gene regulators that function mainly by binding the 3′untranslated regions of specific target messenger
RNA (mRNA) leading to gene inactivation by repression of mRNA transcription or induction of mRNA.
Mature miRNAs are short molecules approximately 22 nucleotides in length. They regulate a wide range
of biological functions from cell proliferation and death to cancer progression. Cellular miRNA expression
levels can be used as biomarkers for the onset of disease states and in gene therapy for genetic disorders.
Methods for detection of miRNA mainly include northern blotting, microarray, polymerase chain reaction
(PCR). This review focuses on the use of electrochemical biosensors for the detection of microRNA.
&2013 Elsevier B.V. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
1.1. RNA ............................... ....................................................... ................... 74
1.2. MicroRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
1.2.1. What is the relation between miRNA and non-coding RNAs?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
1.2.2. How many exactly are miRNA genes in the body? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
1.2.3. What is the role of miRNAs in diseases? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
2. Difficulties in miRNA detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
3. Current detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4. Electrochemical approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
4.1. Nano materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
4.1.1. Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
4.1.2. Nanowires . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
4.2. Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
4.3. Electroactive complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.4. Electrocatalytic oxidation of guanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
References..............................................................................................................82
1. Introduction
A biosensor is a device incorporating a molecular recognition
element associated with a physicochemical transducer. Transducing
systems may be optical, electrochemical, thermometric, piezoelectric,
micromechanical or magnetic. Materials that can be used as recogni-
tion elements consist of proteins, tissues, cells, enzymes, nucleic acids
etc. [1].
1.1. RNA
RNA, Ribonucleic acid, is one of the three major macromole-
cules (along with DNA and proteins) in the body that are essential
to all known forms of life. Like DNA, RNA is made up of a long
Contents lists available at SciVerse ScienceDirect
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0039-9140/$- see front matter &2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.talanta.2013.03.061
n
Corresponding author at: Eletroanalytical Chemistry Research Laboratory,
Department of Analytical Chemistry, Faculty of Chemistry, University of
Mazandaran, Babolsar, Iran. Tel.: +98 112 5342392; fax: +98 112 5342350.
E-mail addresses: ehamidiasl@umz.ac.ir,
ehamidiasl@yahoo.com (E. Hamidi-Asl).
Talanta 115 (2013) 74–83

chain of components called nucleotides. There are several kinds of
RNA in the body: messenger RNA (mRNA), transfer RNA (tRNA),
ribosomal RNA (rRNA), small nuclear RNA (snRNA) and other non-
coding RNAs (ncRNA). The mRNA carries the genetic information
in all organisms for the synthesis of proteins. Protein synthesis is a
universal function whereby mRNA molecules direct the assembly
of proteins on the ribosome. This process needs to tRNA molecules
to deliver amino acids to the ribosome. Then, rRNA links amino
acids together to form proteins. A snRNA is a class of small RNA
molecules that are found within the nucleus of eukaryotic cells.
They are transcribed by RNA polymerase and are involved in a
variety of important processes such as regulation of transcription
factor and maintaining the telomerase. A ncRNA is a RNA molecule
that is not translated into a protein. The number of ncRNAs
encoded within the human genome is unknown; however recent
transcriptomic and bioinformatics studies suggest the existence of
thousands of ncRNAs. Many ncRNAs show abnormal expression
patterns in cancerous tissues [2–4].
1.2. MicroRNA
1.2.1. What is the relation between miRNA and non-coding RNAs?
One category of ncRNAs is MicroRNAs (miRNAs). When the
human genome project mapped its first chromosome in 1999, it
was predicted the genome would contain over 100,000 protein
coding genes. However, only around 20,000 were eventually
identified [5]. Up to now, the appearance of bioinformatics
approaches combined with genome tiling studies examining the
transcriptome, systematic sequencing of full length DNA libraries,
and experimental validation (including the creation of miRNA
derived antisense oligonucleotides called antagomirs) has revealed
that many transcripts are non protein-coding RNA, including
several snRNAs and miRNAs [6].
miRNAs are short ribonucleic acid molecules, 19–25 nucleotides
long and exist in all eukaryotic cells. miRNAs are post-
transcriptional regulators that bind to complementary sequences
on target messenger RNA transcripts. Often, they result in transla-
tional repression and gene silencing. The human genome may
encode over 1000 miRNAs, which may target about 60% of
mammalian genes and are abundant in many human cell types
[7]. The first miRNAs were inadvertently discovered in 1993 by
Ambros et al. during a study of the lin-14 gene in C. elegans
development [8] but until the early 2000s, miRNAs were not
recognized as a distinct class of biological regulators with con-
served functions. Most miRNA genes are found in intergenic
regions or in the anti-sense orientation of genes and contain their
own miRNA gene promoter and regulatory units. Molecular
biology of miRNA, molecular mechanisms of miRNA action, meth-
odologies and normal physiological functions of miRNAs are
described very well in a review by Quesne and Caldas [9]. To date
more than 5000 miRNAs have been annotated in vertebrates,
invertebrates and plants according to the miRBase database
(http://microrna.sanger.ac.uk/).
1.2.2. How many exactly are miRNA genes in the body?
The number of miRNA hairpin loci in the miRBase database
continues to increase quickly, from 2909 in 36 genomes (year
2005) to 5071 in 58 genomes (year 2007). The number of miRNAs
in a genome has been the topic of much investigation in the
literature. Early estimates of the number of miRNAs in the worm
and human genomes were put at 123 and 255, respectively [10,11].
However, these estimates were based largely on conservation
studies. Recently, a number of huge studies have lifted the number
of miRNA loci known in human, around 60% of which are
conserved in mouse [12].
1.2.3. What is the role of miRNAs in diseases?
So far, it has been found that several miRNAs associate with
diseases such as some types of cancers, heart diseases, diabetes,
nervous system, kidney and liver diseases [13]. Numerous studies
have reported that miRNAs are either increased (upregulated) or
decreased (downregulated) in cancer cells [14].
miRNAs have an effect on the development of cancer. Mice that
were engineered to produce a surplus of types of miRNAs found in
lymphoma cells developed the disease within 50 days and died
two weeks later. However, mice without the miRNA surplus lived
over 100 days [15]. Leukemia can be caused by the insertion of a
viral genome into the mir–17 –92 cluster gene leading to enhanced
expression of mature miRNAs derived from this cluster [16].
Another example is the study of O'Donnell et al. [17]. They found
that two types of miRNAs inhibit the E2F1 protein, which regulates
cell proliferation. miRNAs appear to bind to messenger RNA before
it can be translated into proteins that switch genes on and off. By
measuring activity among 217 genes encoding miRNA, patterns of
gene activity that can distinguish types of cancers can be dis-
cerned. miRNA signatures may enable classification of cancer. This
will allow doctors to determine the original tissue type which
spawned a cancer and to be able to target a treatment course
based on the original tissue type. Nielsen et al. [18] developed a
novel miRNA-profiling based screening assay for the detection of
early-stage colorectal cancer which is currently in clinical trials.
Early results showed that blood plasma samples collected from
patients with early, resectable (Stage II) colorectal cancer could be
distinguished from those of sex-and age-matched healthy volun-
teers. Sufficient selectivity and specificity could be achieved using
small (less than 1 ml) samples of blood. The test has potential to be
a cost-effective, non-invasive way to identify at-risk patients who
should undergo colonoscopy.
A study about miRNA and heart diseases by Zhao et al. [19]
shows the role of miRNA function in the heart. It has been
addressed by conditionally inhibiting miRNA maturation in the
murine heart, and has revealed that miRNAs play an essential role
during its development. miRNA expression profiling studies
demonstrate that expression levels of specific miRNAs change in
diseased human hearts, pointing to their involvement in cardio-
myopathies. Furthermore, studies on specific miRNAs in animal
models have identified distinct roles for miRNAs both during heart
development and under pathological conditions, including the
regulation of key factors important for cardiogenesis, the hyper-
trophic growth response, and cardiac conductance [20,21].
Schratt in his study [22] illustrated that miRNAs regulate the
nervous system. Neural miRNAs are involved at various stages of
synaptic development, including dendritogenesis, synapse forma-
tion and synapse maturation [23].
2. Difficulties in miRNA detection
Cissell et al. described the challenges about miRNA detection in a
review [24].Asbriefly, the problems are small size of miRNA and
sensitivity of the assay. The concentration of cellular miRNA is around
1000 molecules per cell. The overall assay time will be increased, if
the assay needs isolation of miRNA. For in situ detection, when a
sample is a mixture of pre- and mature miRNA, the oligonucleotide
probecanhybridizenonspecifically to pre-miRNA. This can cause
false positive signal for expression levels of mature miRNA.
3. Current detection methods
The most widely used miRNA detection methods are northern
blotting. When the first miRNAs were reported, northern blotting
E. Hamidi-Asl et al. / Talanta 115 (2013) 74–83 75

was applied to detect these small RNAs [25]. Northern blotting
involves the use of electrophoresis to separate RNA samples by
size and detection with a hybridization probe complementary to
part of or the entire target sequence [26–31]. The second most
widely used technique is cloning. The cloning of miRNA was one of
the first techniques and the main tool used to identify miRNAs
[32–37]. Sequencing is also frequently used [35]. The other one is
microarray that consists of an arrayed series of thousands of
microscopic spots of DNA oligonucleotides each containing of
10
−12
mol of a probe. Probe-target hybridization is usually detected
and quantified by detection of the fluorophore, silver, or chemilu-
minescence labelled targets to determine relative abundance of
nucleic acid sequences [38–44].Reverse transcriptase polymerase
chain reaction (RT-PCR) is a conventional miRNA detection method
in the biomedical field that can detect miRNA in real time. However,
the most important issue concerning conventional RT-PCR is the
fact that it is a semi-quantitative technique [45–48]. Some other
techniques have been reported for the detection of miRNAs. Surface-
enhanced Raman scattering (SERS) is a label-free way [49–53].The
method of Surface plasmon resonance (SPR) has been reported for
rapid and sensitive miRNA detection in less than 30 min at
concentrations down to 2 pM [54–56].Surface plasmon resonance
imaging (SPRi) is a promising technology for the multiplexed
detection of miRNAs. SPR is hybridization-based, label-free method
of identifying RNA and generally based on intensity modulation,
measuring the reflectivity of monochromatic incident p-polarised
light at a fixed angle [57–59]. Various papers have been published to
detect miRNA in different cancers using Fluorescence methods [60–
63].Bioluminescence method offers detection limits in the picomole
to femtomole range, which are sensitive enough for expression level
studies in total cellular extract and demonstrates selectivity for the
target small molecule in a total RNA sample, which simplifies the
assays by removing isolation and amplification steps [64–67].
4. Electrochemical approaches
Electrochemical genosensors hold great promise to serve as
devices suitable for point-of-care diagnostics and multiplexed
platforms for fast, simple and inexpensive nucleic acids analysis.
A typical electrochemical biosensor is made of a solid electrode
with immobilized short single-stranded nucleotide probe on it and
electroactive hybridization indicators. The hybridization between
the probe and the complementary sequence influences the per-
formance of electrochemical biosensors.
All of the electrochemical miRNA detection methods have been
presented in the literature relying on hybridization. Once hybridi-
zation occurs, there must be a way to translate the hybridization
event into a measurable signal. One of the limiting factors for the
development of electrochemical genosensors is the sensitivity.
When the specific target gene is present as a single copy in the
organism genome, the amount of DNA/RNA that has to be detected
is at the attomolar to femtomolar level [68,69]. So far, several
approaches have been reported for signal amplification and
further improvement the sensitivity of the analytical protocols.
As below, can be seen the used methodology for this purpose.
4.1. Nano materials
4.1.1. Nanoparticles
Gao and Yang reported detection of miRNAs using electrocatalytic
nanoparticle tags [70]. They used an indium tin oxide electrode on
which oligonucleotide capture probes are immobilized. After hybri-
dization with periodate-treated miRNA, the nanoparticle tags,
isoniazid-capped OsO
2
nanoparticles, were brought to the electrode
through a condensation reaction to chemically amplify the signal. The
nanoparticles effectively catalyse the oxidation of hydrazine and
greatly enhance the detectability of miRNAs. Fig. 1 shows the
schematic of miRNA assay using electrocatalytic OsO
2
nanoparticles.
In another paper, Gao and Yu presented a chemically amplified
approach that allows the direct detection of specific miRNAs in
total RNA. Prior to the test, miRNA molecules were directly tagged
with an electrocatalytic moiety under mild conditions. In the
presence of ascorbic acid (AA), the current generated from the
electrocatalytic oxidation of AA was detected amperometrically
and it correlated directly to the concentration miRNA in the
sample solution [71].
Recently, Peng and Gao reported an amplified detection of
miRNA based on ruthenium oxide nanoparticle-initiated deposi-
tion of an insulating film [72]. They presented an electrochemical
impedimetry biosensor along with a novel sensing protocol that
enabled amplified electrical detection of miRNA with significantly
enhanced specificity and sensitivity. The biosensor was based on a
direct ligation procedure that involved a chemical coupling reac-
tion to directly tag miRNAs with ruthenium oxide nanoparticles
(RuO
2
NPs). The RuO
2
NPs effectively catalysed the polymerization
of 3,3′-dimethoxybenzidine (DB), and the hybridized miRNA
strands and free capture probe strands guided the deposition of
poly(3,3′-dimethoxybenzidine) (PDB). The amount of the depos-
ited PDB and its insulating power directly correlated to the
concentration of the target miRNA in the solution. There was no
cross-hybridization between pre-miRNA and mature miRNA and
very little cross-hybridization among closely related miRNA family
members even at single-base-mismatched levels. The nature of
the protocol greatly enhanced the sensitivity of the biosensor and
may enable the development of a portable multiplexing miRNA
profiling system. Fig. 2 illustrates scheme of the miRNA biosensor
based on RuO
2
NP.
Dong et al. introduced a simple, sensitive, and label-free
method for miRNA biosensing using oligonucleotide encapsulated
silver nanoclusters (Ag-NCs) as effective electrochemical probes
[73].Fig. 3 depicts the basis of their approach. The functional
oligonucleotide probe integrates both recognition sequence for
hybridization and template sequence for in situ synthesis of
Ag-NCs, which appears to possess exceptional metal mimic
enzyme properties for catalysing H
2
O
2
reduction. The gold elec-
trodes were employed to immobilize the molecular beacon (MB)
probe. After the MB probe subsequently hybridizes with the target
and functional probe, the oligonucleotide encapsulated Ag-NCs are
brought to the electrode surface and produce a detection signal, in
response to H
2
O
2
reduction.
4.1.2. Nanowires
Nanowires have many interesting properties that are not seen
in bulk or 3-D materials. This is because electrons in nanowires are
Fig. 1. Schematic illustration of miRNA assay using electrocatalytic OsO
2
nanopar-
ticles Ref. [70].
E. Hamidi-Asl et al. / Talanta 115 (2013) 74–8376

quantum confined laterally and thus occupy energy levels that are
different from the traditional continuum of energy levels or bands
found in bulk materials [74].
Zhang et al. reported a label-free direct detection of miRNAs with
silicon nanowire biosensors [75]. They represented a label-free and
direct hybridization assay for ultrasensitive detection of miRNA using
silicon nanowires (SiNWs) device. Peptide nucleic acids (PNAs), which
serve as a receptor to recognize miRNA directly without labelling the
target miRNA, were immobilized on the surface of the SiNW device.
Electrical measurements for the sensing experiments were carried out
by detecting the resistance change of SiNWs before and after PNA–
miRNA hybridization. Schematic illustration of this device is shown in
Fig. 4. The device enables identification of fully matched versus
mismatched miRNA sequences and is capable of detecting miRNA in
total RNA extracted from Hela cells. Sensitivity of the SiNW biosensors
is affected by a number of issues such as NW size, surface chemistry,
Debye length, and charge large distance. Moreover, hybridization
conditions including ionic strength, temperature and time are critical
for hybridization efficiency, thereby affecting detection sensitivity.
Higher thermal stability and melting temperature of the PNA–RNA
duplex than that of a PNA–DNA duplex indicates that PNA has higher
affinity for RNA than for DNA, and PNA/RNA duplex is more stable than
a PNA/DNA duplex. This may induce higher detection sensitivity for
detection of RNA than DNA using the PNA-functionalized SiNW device.
Fan et al. introduced a detection of miRNAs using target-guided
formation of conducting polymer nanowires in nanogaps [76].
Hybridization and electrical detection of the sensing procedure
was depicted in Fig. 5. In their study peptide nucleic acid (PNA)
was used as capture probes and polyaniline (PAn) nanowires as
conducting polymer nanowires. The PNA was immobilized in
nanogaps of a pair of interdigitated microelectrodes and hybridi-
zation was performed with their complementary target miRNA.
Then, deposition of (PAn) nanowires was carried out by an
enzymatically catalysed method, where the electrostatic interac-
tion between anionic phosphate groups in miRNA and cationic
aniline molecules was exploited to guide the formation of the PAn
nanowires onto the hybridized target miRNA. The conductance of
the deposited PAn nanowires correlates directly to the amount of
the hybridized miRNA. This approach directly utilized chemical
ligation and amplification for signal read-out and thus eliminated
the use of labelling probes, which greatly simplifies the detection
procedure. A much lower detection limit could be realized with
longer target nucleic acids, because the bridging of the nanogaps
by the PAn nanowires can be realized with fewer long nucleic acid
molecules. The dynamic range for the biosensor was from 10 fM to
20 pM with a detection limit of 5.0 fM.
4.2. Enzymes
The successful and widespread use of enzymes as labels in
affinity bioassays is essentially due to their ability to convert single
hybridisation events into a multitude of detectable molecules.
Recently, several papers report the use of enzymatic reaction for
recognition of a hybridization event between probe and miRNA
targets.
In this way, Pohlmann and Sprinzl reported an electrochemical
detection of miRNAs via gap hybridization assay [77]. They
introduced a method for detection of mature miRNAs based on
four components DNA/RNA hybridization and electrochemical
detection using esterase 2-oligodeoxynucleotide (EST2-ODN)
conjugates. Due to complementary binding of miRNA to a gap
built of capture and detector oligodeoxynucleotide, the reporter
enzyme is brought to the vicinity of the electrode and produces
enzymatically an electrochemical signal. The schematic principle
of miRNA detection is shown in Fig. 6. The key feature of this
method is generation of a continuous base stacking between
immobilized capture ODN, miRNA, EST2-ODN conjugate, and
complementary RNA probe. EST2 as a reporter enzyme is superior
to most other reporter enzymes because of the possibility to
Fig. 2. Schematic illustration of the miRNA biosensor based on RuO
2
NP-catalyzed miRNA-templated deposition of a thin PBD insulating film Ref. [72].
Fig. 3. Illustration of electrochemical detection of miRNA using Oligonucleotide
Encapsulated Ag-NCs Ref. [73].
Fig. 4. Schematic illustration of the label-free direct hybridization assay developed
for ultrasensitive detection of miRNA Ref. [75].
E. Hamidi-Asl et al. / Talanta 115 (2013) 74–83 77

site-specific modification with ODNs. Furthermore, EST2 is a
thermostable enzyme enabling the reporter enzyme to be present
during hybridization reactions at elevated temperatures. In the
absence of miRNA, the gap between capture and detector oligo-
deoxynucleotide is not filled, and missing base stacking energy
destabilizes the hybridization complex. The gap hybridization
assay demonstrates selective detection of miR-16 within a mixture
of other miRNAs, including the feasibility of single mismatch
discrimination.
Cai et al. reported an electrochemical sensor based on label-free
functional allosteric molecular beacons for detection target DNA/
miRNA [78]. Their sensors were fabricated using label-free functional
allosteric molecular beacons (aMBs), which can form streptavidin
aptamers to bind to streptavidin peroxidase polymer and so generate
Fig. 5. Schematic illustration of the sensing mechanism Ref. [76].
Fig. 6. Electrochemical detection of gap hybridization among immobilized capture ODN (red), miRNA (green), EST2-miR conjugate (yellow), and complementary RNA probe
(blue) Ref. [77]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
E. Hamidi-Asl et al. / Talanta 115 (2013) 74–8378

catalytic currents in the presence of the targets. These sensors
eliminate the antigen antibody interactions which require sophisti-
cated DNA modification. Fig. 7 illustrates the principle of the RSV-
aMB E-sensor for RSV DNA detection. This aMB contained a sequence
of SA aptamer that was blocked by the hairpin structure. When the
aMBhybridizedwiththetargetDNA(ormiRNA),theSAaptamer
formed a special structure that bound to SA-HRP protein to generate
catalytic electrochemical signal. One positively charged TMB
+
,an
oxidization product of TMB, could be adsorbed to the negatively
charged DNA backbone, resulting in a pair of adsorption and
desorption waves in the redox reaction process in the cyclic
voltammogram. Another pair of waves was observed when the
aMB E-sensor was hybridized to the perfect complement of the
aMB sequence, and this contributed to the redox reaction between
TMB
+
and TMB
2+
and the intercalation process of the TMB
2+
to
dsDNA. Amperometry was used to measure the catalytic current. The
aMB E-sensors showed very good sensitivity and selectivity to the
targets, and reached detection limits of 44 amol RSV DNA and
13.6 amol miRNA let-7a in the 4 mL sample. In addition, the RSV
aMB E-sensor could perform well in 10% serum samples.
Bettazzi et al. reported an electrochemical detection of miRNA-
222 by use of a magnetic bead-based bioassay and enzyme
amplification [79]. The proposed bioassay was based on biotiny-
lated DNA capture probes immobilized on streptavidin-coated
paramagnetic beads. Total RNA was extracted from the cell sample,
enriched for small RNA, biotinylated, and then hybridized with the
capture probe on the beads. The beads were then incubated with
streptavidin–alkaline phosphatase and exposed to the appropriate
enzymatic substrate. The product of the enzymatic reaction was
electrochemically monitored. The assay was finally tested with a
compact microfluidic device which enables multiplexed analysis of
eight different samples with a detection limit of 7 pmol L
−1
and
RSD¼15%. In this paper, application of the method to real samples
is presented (Fig. 8).
Yin et al. [80] described a sensitive and selective miRNA
biosensor based on graphene and dendritic gold nanostructure
(DenAu) modified glassy carbon electrode (GCE), LNA integrated
molecular beacon probe, multifunctional encoded DNA–AuNPs–
LNA bio bar codes and HRP catalysis signal amplification, in which,
the molecular beacon was not labelled with fluorescent and
quencher groups, only a –SH was labelled at its 5′-end for assembly
on the electrode surface. The AuNPs were labelled with two
oligonucleotide strands. One is LNA integrated DNA, which was
complementary to the 3′-end of the molecular beacon probe. The
other is biotin functionalized DNA, not complementary to the
probe. The length of the probe (41 bases) was longer than the
target miRNA (22 bases). The 5′-end of the molecule was com-
plementary to miRNA and its 3′end was complementary to LNA
integrated DNA in DNA–AuNPs–LNA bio bar codes. After hybridiza-
tion with target miRNA, the stem-loop structure of the molecular
beacon was unfolded and its 3′-end was forced far away from the
electrode surface to hybridize with LNA integrated DNA. Then,
with specific interaction between biotin and streptavidin, the
streptavidin functionalised horseradish peroxidase (streptavidin–
HRP) can be immobilized on the electrode surface to catalyse the
oxidation reaction of hydroquinone by H
2
O
2
to form benzoquinone
and enhance the electrochemical reduction signal of benzoqui-
none. The detection strategy was shown in Fig. 9. Taking advan-
tage of tri-amplification effects of the DenAu/graphene/GCE,
multifunctional encoded AuNP and HRP, the biosensor showed
high determination sensitivity for miRNA with a detection limit of
0.06 pM.
Kilic et al. reported an electrochemical based detection of
mir21 in breast cancer cells [81]. In their study, the oxidation
signal of enzymatic reaction product, alpha naphtol (a-NAP),
which is expected to be produced in the presence of hybrid,
was detected by differential pulse voltammetry on a disposable
PGE. The steps of the method are shown in Fig. 10. They claimed
that for the first time an enzyme based biosensor was designed
for the detection of miRNA from cell lysates without any
modification of the sample. As a positive control, total RNA
isolated from a breast cancer cell line that contains unregulated
mir21 was used. The specificity of the assay was proved by non-
complementary studies using mir21 free total RNA samples. The
proposed enzyme based assay seems to provide more reprodu-
cible results for the detection of miRNA than conventional
guanine oxidation based method for cell lysates. Additionally,
the method can unambiguously distinguish between mir21
including samples and mir21 free samples while detection is
impossible with the guanine based method due to very low
signal.
4.3. Electroactive complexes
The first electroactive label of nucleic acids based on osmium
tetroxide complexes with nitrogenous ligands was invented by
Palecek et al. in 1981 [82]. These complexes bind covalently to
pyrimidine bases through addition to the 5,6 double bond in
single stranded or distorted double stranded DNA structures.
Trefulka et al. reported the facile end-labelling of RNA with
electroactive Os(VI) complexes [83]. They showed that ribose at
the oligo 3′-end can be selectively labelled by Os(VI)2,2′-bipyridine
Fig. 7. The principle of the RSV-aMB E-sensor for RSV DNA detection Ref. [78].
E. Hamidi-Asl et al. / Talanta 115 (2013) 74–83 79

(bipy) yielding an electroactive adduct detectable at carbon and
mercury electrodes. Exchanging ligands in Os(VI) complexes offer
interesting possibilities in changing the chemical and
electrochemical properties of the RNA adducts. They used square
wave voltammetry and could detect 22-mer oligos down to
250 nM. The Os(VI)bipy-oligo adducts produced an electrocatalytic
Fig. 8. Schematic diagram of the assay: (a) biotinylated DNA capture probes, (b) cell samples are lyzed in a denaturing lysis solution, (c) probe-modified and biotin-blocked
beads are incubated with the biotinylated miRNA targets, (d) the biotinylated hybrids are labeled with a streptavidin–alkaline phosphatase conjugate, (e) the assay has also
been integrated into a microfluidic device, (f) schematic diagram of beads blocked within the microchannel, on the electrode surface, by the magnet Ref. [79].
Fig. 9. Chronoamperometry determination of miRNA hybridization through three steps of amplification Ref. [80].
E. Hamidi-Asl et al. / Talanta 115 (2013) 74–8380

peak near −1.2 V at mercury electrodes allowing their determina-
tion down to picomolar concentrations. High specificity of Os(VI)
bipy for ribose in nucleic acids and high sensitivity of the
determination at mercury and solid amalgam electrodes give
promise for new efficient methods of miRNA determination.
Gao and Yu reported a direct labelling miRNA with an electro-
catalytic moiety and its application in ultrasensitive miRNA assays
[84]. In this paper, miRNA is directly labelled with Ru(PD)
2
Cl
2
(PD¼1,10-phenanthroline-5,6-dione), through coordinative bonds
with purine bases in the miRNA molecule. The schematic of assay
illustrated in Fig. 11. The protocol for hybridization and detection
was as follows: first, the electrode was placed in a moisture
saturated environmental chamber maintained at 30 1C. A 2.5 μl
aliquot of hybridization solution, containing the desired amount of
labelled miRNA, was uniformly spread onto the electrode. It was
then rinsed thoroughly with a blank hybridization solution at
30 1C after a 60-min of hybridization period. The hydrazine
electrooxidation current was measured amperometrically at
0.10 V in vigorously stirred PBS containing 5.0 mM hydrazine. At
low miRNA concentrations, smoothing was applied after each
amperometric measurement to remove random noise and electro-
magnetic interference. The amplification from the electrocatalytic
oxidation of hydrazine greatly enhances the detectability of the
approach.
4.4. Electrocatalytic oxidation of guanine
The label-free monitoring of electrochemical DNA or RNA
hybridization detection, based on guanine moiety oxidation signal
of the probe or target on conventional electrodes in electroche-
mical biosensor, seems to be a simple, less time consuming and
more applicable strategy in comparison with the others. In the
most label free DNA biosensors, an inosine-substituted oligonu-
cleotide is used as a probe. It means that there is not guanine
in the probe sequence, instead there are several cytosines. In
this way, it can be obtained an enhanced electrochemical signal
during hybridization with target containing several guanine bases
[85,86].
An innovative electrochemical approach for an early detection
of miRNAs was reported by Lusi et al. [87]. They presented an
electrochemical genosensor, based on guanine oxidation conse-
quent to the hybrid formation between the miRNA and its inosine
substitute capture probe. The oxidation of guanine during the
hybrid formation on the electrode surface generates an electrical
signal that is evaluated by a differential pulse voltammetry
technique. Fig. 12 illustrates the scheme of this assay. This
electrochemical determination is label free and does not require
a direct miRNA labelling with toxic substances. However, the
sensitivity of the assay was poor.
Fig. 10. Sketch of steps involved in (A) surface activation of PGE with NHS/EDC, (B) probe immobilization on to the surface of PGE, (C) hybridization of probe with
biotinylated target, (D) Ex-Ap–biotin interaction, (E) enzyme substrate interaction and production of α-NAP and (F) voltammetric measurement of oxidation signal of α-NAP
Ref. [81].
Fig. 11. Schematic representation of a miRNA assay using electrocatalytic label Ref. [84].
E. Hamidi-Asl et al. / Talanta 115 (2013) 74–83 81

5. Conclusions
Deoxyribonucleic acid is the carrier of genetic information and
the foundation material of biological heredity. The attempt of
biotechnology is rapid DNA sequencing. Therefore, sequence infor-
mation is now available for many diseases. To more effectively
combat these diseases in the medical arena and accelerate response
to bioterrorism threats, early and accurate detection of DNA markers
is crucial. In this area, multidisciplinary teams of researchers
including chemists, biochemists, and physicists have done lots of
work to simple routine DNA detection. MiRNAs are small noncoding
RNA gene products about 22 nucleotides long that are found in
diverse organisms, including animals and plants. The mature miR-
NAs control gene expression interacting with a specific mRNA, either
inducing its degradation or blocking the translation process. It is
now predicted that as much as 40–50% of mammalian mRNA could
be regulated at the translational level by miRNAs and they have a
greatimpactinbiologicalprocesses.Therefore,thereisanurgent
need to reliable and ultrasensitive test for miRNAs. At the present
time, miRNAs are detected with techniques such as Northern blot,
RT-PCR, and microarrays using many commercial kits. Because the
inherent superiorities of electrochemical transduction methods such
as excellent compatibility with advanced semiconductor technology,
miniaturization and low cost, nucleic acid biosensors based on
electrochemical detection are able to provide high performance at
low cost with simple miniaturised redout, and thus are exempt from
the problems encountered in the other detection systems [88].
In this review the rapid progresses of miRNA electrochemical
sensors were highlighted and the elegant sensing concepts were
summarised. These approaches may have influence upon detection
of pathogens, genetic mutations and targets of pharmacogenomics
and industrial interest in the near future. Industrial development of
electrochemical methods needs to increase in automation, transduc-
tion of the hybridization reaction into a measurable electrochemical
signal and performance of the sensor with real biological samples.
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