sensors

Review
Label-Free Aptasensors for the Detection of Mycotoxins
Amina Rhouati 1,2 , Gaelle Catanante 1 , Gilvanda Nunes 3 , Akhtar Hayat 1,4, * and
Jean-Louis Marty 1, *
1 BAE Laboratory, Universit de Perpignan Via Domitia, 52 Avenue Paul Alduy, Perpignan 66860, France;
a.rhouati@ensbiotech.edu.dz (A.R.); gaelle.catanante@univ-perp.fr (G.C.)
2 Ecole Nationale Suprieure de Biotechnologie, Constantine 25100, Algeria
3 Technological Chemistry Department, Federal University of Maranho, CCET/UFMA, Av. Portugueses,
Cidade Universitria do Canga, 65080-040 So Luis, Brazil; gilvanda.nunes@hotmail.com
4 Interdisciplinary Research Centre in Biomedical Materials (IRCBM) COMSATS Institute of Information
Technology (CIIT), Lahore 54000, Pakistan
* Correspondence: akhtarhayat@ciitlahore.edu.pk (A.H.); jlmarty@univ-perp.fr (J.-L.M.);
Tel.: +33-468-66-2257 (J.-L.M.)

Academic Editor: Beate Strehlitz

Received: 31 October 2016; Accepted: 14 December 2016; Published: 18 December 2016

Abstract: Various methodologies have been reported in the literature for the qualitative and
quantitative monitoring of mycotoxins in food and feed samples. Based on their enhanced specificity,
selectivity and versatility, bio-affinity assays have inspired many researchers to develop sensors by
exploring bio-recognition phenomena. However, a significant problem in the fabrication of these
devices is that most of the biomolecules do not generate an easily measurable signal upon binding to
the target analytes, and signal-generating labels are required to perform the measurements. In this
context, aptamers have been emerged as a potential and attractive bio-recognition element to design
label-free aptasensors for various target analytes. Contrary to other bioreceptor-based approaches,
the aptamer-based assays rely on antigen binding-induced conformational changes or oligomerization
states rather than binding-assisted changes in adsorbed mass or charge. This review will focus on
current designs in label-free conformational switchable design strategies, with a particular focus on
applications in the detection of mycotoxins.

Keywords: aptamer; label free detection; mycotoxins; conformational changes; current trends

1. Introduction
Biosensors have emerged as a cheap and quick alternative to traditional chromatographic
methods in the analytical assayfield. Biosensors are analytical tools which rely on the integration of
bio-recognition molecules in the construction of sensor design. The most commonly employed
bio-receptor elements in the biosensor application domain include enzymes, antibodies and
aptamers [1,2]. However, the enzymatic biosensor field suffers from various drawbacks and their
real time use is limited to certain specific applications. Several factors such as conformational change
in amino acids at the active site of the enzyme can induce dramatic changes in enzymatic activity
and substrate specificity, and subsequently influence the stability of the sensing enzymatic reaction.
Enzyme denaturation is a property of vital significance in the fabrication of enzymatic biosensors.
Enzyme denaturation can be induced by changes in pH, temperature, pressure, exposure to UV
radiation, detergents, organic solvents or certain chemicals. The enzyme isolation process and
subsequent incorporation into an in vitro operating environment can result in a loss of the enzymatic
activity [3]. Though every enzyme has a set of specific optimum working conditions, but still a lot of
research has been focused on how to improve the enzymatic stability over a long period of time [4].
As an alternative to enzymatic assays, one of the attractive candidates is immunoassays, likely due

Sensors 2016, 16, 2178; doi:10.3390/s16122178 www.mdpi.com/journal/sensors

Sensors 2016, 16, 2178 2 of 21

to the high affinity interactions between antigens and antibodies, often allowing higher sensitivity
and lower limits of detection. However, antibodies are also proteinic in nature, and are prone to
denaturation phenomena under varying experimental and physiological conditions. Moreover, they are
mainly produced in living animals, and this results in relatively very high assay costs [5]. Phage display
technology is proposed for in vitro selection of monoclonal antibodies characterized by high specificity
and affinity. This technique is based on the genetic engineering of bacteriophages and repeated rounds
of antigen-guided selection and phage propagation. However, heavy and light chain pairing may not
reflect that of in vivo immunoglobulin [6]. In this context, a new class of molecules, named aptamers,
has appeared as promising recognition tools for analytical applications. Aptamers are short single
stranded oligonucleotides, either DNA or RNA, that fold into well-defined 3D structures and bind
to their ligand by complementary shape interactions, with antibody-like binding ability. Aptamers
present significant advantages over antibodies. As they are chemically synthesized, their production
does not require the use of animals and is therefore less expensive and tedious. Aptamers can be
also easily labeled with a wide range of reporter molecules such as fluorescent dyes, enzymes, biotin,
or aminated compounds, enabling the design of a variety of detection methods [7]. Furthermore,
the function of immobilized aptamers can be easily regenerated and aptamers can be reused.
Due to these advantages, aptamers can thus be considered as a valid alternative to antibodies or
other bio-receptors, in developing various analytical techniques. Various format assays using aptamers
as bio-recognition elements have been reported in the literature. The conventional enzyme-linked
aptamer assays, however, are time consuming, expensive and involve multistep processes.
As aptamers and antigens are chemically inert, a label such as an enzyme is required to generate an
electrochemical signal [68]. Although these developed methods are very sensitive, however, there are
several challenges in the construction of labeled aptasensors. Labeling of either antigen or aptamer
makes the assays more complex, time consuming and laborious. Moreover, the labeling process is
costly and often results in the denaturation of the modified biomolecules [9]. In an effort to overcome
these drawbacks, there is increasing interest in the development of label-free aptasensors.
On the other hand, since many small target analytes such as mycotoxins are present at
low-levels, there are increasing demands for ultrasensitive detection methods for such molecules
in the agro-food domain. High sensitivity is required because of the low maximum permissible levels
of mycotoxins established by the European Commission. For example, ochratoxin A amount should not
exceed 2 g/Kg in wines and coffee and 5 g/Kg in cereal products [8], while the limits for aflatoxin B1
in different foodstuffs have been set between 0.05 and 20 g/Kg [9]. However, it is difficult to achive
ultrasensitive detection of small molecules by a basic aptasensor design because of the very small size of
the analytes. This problem has resulted in the exploration of novel aptasensor methods and designs to
carry out the sensitive detection of mycotoxin analytes. Many methods for signal amplification
in aptasensor design have been demonstrated, such as rolling circle amplification [10], strand
displacement amplification [11] and enzyme label [12]. Although these methods offer advantages
in terms of signal amplification, but are complicated, expensive and their operating conditions are
rigorous [13]. Besides, aptamer-functionalized nanoparticles have been reported to amplify the
signal in the development of aptasensors [14]. Using nanoparticles might overcome some of the
problems, but still sensitive and in consistent methods are strongly desirable in the development of
aptasensors. Based on the above observations, the objective of this review paper was to compare
label and label-free methodologies, formats and detection techniques in the label-free aptasensor field,
and finally mycotoxin detection as an example of small molecule analysis based on the label-free
aptasensing methodologies is described in detail.
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2016, 16, 2178 3 of 21

2.2.Why
WhyLabel-Free
Label-FreeDetection?
Detection?

2.1.
2.1.Labeled
Labeledvs.Unlabeled
vs.UnlabeledScreening
ScreeningofofBiomolecular
BiomolecularInteractions
Interactions
The
Theuseuse of labels
labelstotoreport
report bio-recognition
bio-recognition eventsevents is very
is very common common in the development
in the development of
of detection
detection
strategiesstrategies
employed employed
in differentin different
fields. The fields. Theofnature
nature the usedof the used molecule
molecule can varycan vary widely;
widely; the label
the
canlabel can be a radioactive
be a radioactive or a fluorescent
or a fluorescent dye [15,16], dye metal [15,16],
complex metal complex or nanoparticles
or nanoparticles [17], enzyme with [17],a
enzyme with a detectable product etc [18]. This label is routinely attached to
detectable product . . . etc. [18]. This label is routinely attached to the target molecule or the bioreceptor the target molecule
or(For
theexample:
bioreceptor (For example:
aptamer or antibody).aptamer or antibody).
Analysis Analysisby
is then achieved is measuring
then achieved the by measuring
label activity or the
by
label activity or by the change in other chemical or physical properties on
the change in other chemical or physical properties on the transducer surface. The easy conjugation the transducer surface. The
easy conjugationdetection
and convenient and convenient
are the most detection
importantare characteristics
the most important characteristics
of a marker molecule. Theseof a marker
features
molecule.
are requiredThese features
to ensure are amplification
signal required to ensure signal amplification
for enhancing the sensitivityfor andenhancing
selectivitythe of sensitivity
the method.
and selectivity
Despite of the method.
the advantages Despite the advantages
of label-dependant technology, of the
label-dependant
labeling and technology,
immobilization the labeling
steps are
and
timeimmobilization
consuming and steps are time Indeed,
expensive. consuming and expensive.
non-specific Indeed,
adsorptions andnon-specific
luck of affinityadsorptions
between and
the
luck of affinity
labeled receptorbetween the labeled
and its target could be receptor
observed.andThisits target
changecouldin thebe observed.
binding This change
properties in the
can reduce the
binding properties can reduce the reproducibility, sensitivity or selectivity
reproducibility, sensitivity or selectivity of the biosensor [19]. In addition to these drawbacks, online of the biosensor [19]. In
addition
monitoringto these
is notdrawbacks,
possible with online monitoring
labeled detection is not possible with labeled detection methods.
methods.
Recently,
Recently, label-free technology has emerged asan
label-free technology has emerged as animportant
importantstrategy
strategytotoallow allowthe thestudy
studyofof
biomolecular interactions in real time. By avoiding the laborious labeling
biomolecular interactions in real time. By avoiding the laborious labeling steps and the challenging steps and the challenging
label
labelreaction/operations,
reaction/operations, the
thecost
costofofthethebiosensor
biosensorisisreduced
reducedand andthe theanalysis
analysiscan canbebeperformed
performed
within
within a shorter time. In this kind of operation, both partner molecules (target and bioreceptor)are
a shorter time. In this kind of operation, both partner molecules (target and bioreceptor) are
not
not modified;
modified; theytheyare areusedused in their
in their natural
natural form.form. The bioreceptor
The bioreceptor is immobilized
is immobilized onto the
onto the transducer
transducer
surface, and surface,
the sample and containing
the sample the containing the target
target is directly incubated is with
directly incubated with
the functionalized the
surface.
functionalized surface. Analysis is then performed by studying the change
Analysis is then performed by studying the change in electrical or physical properties of the surface in electrical or physical
properties
which dependof thesolely
surface onwhich depend
the affinity solely on the
of interaction affinitythe
between of interaction
analyte andbetweenits receptorthe analyte
and thus and
the
its receptor and thus the concentration of the analyte in the sample. The use
concentration of the analyte in the sample. The use of label-free monitoring increases the retention of of label-free monitoring
increases the retention
the high affinity of the high
and decreases affinity adsorptions
non-specific and decreases [20].non-specific
Figure 1 shows adsorptions
the principal[20].differences
Figure 1
shows
betweenthe labeled
principal anddifferences
non-labeled between labeled and non-labeled biosensors.
biosensors.

Labelfree
Figure1.1.Label
Figure free(a) vs.label
(a)vs. labeldependent
dependentbiosensors
biosensors(b).
(b).

2.2.Label
2.2. LabelFree
FreeDetection
DetectionMechanism
Mechanism
InIncontrast
contrasttotoconventional
conventionalbiosensors,
biosensors,constituted
constitutedofofthree
threeessential
essentialpartsa
partsabioreceptor,
bioreceptor,aa
signal reporter and
signal anda measurement
a measurementinstrumentlabel-free biosensors
instrumentlabel-free are only
biosensors arebased
onlyonbased
a bioreceptor
on a
and a physicochemical detector of the surface property changes. For that, the bioreceptor
bioreceptor and a physicochemical detector of the surface property changes. For that, the bioreceptoris first
is first immobilized on a transducer surface, whose properties will be altered upon binding of the
analyte. It is noteworthy that the affinity of the recognition element influences the biosensors
Sensors 2016, 16, 2178 4 of 21

immobilized on a transducer surface, whose properties will be altered upon binding of the analyte.
It is noteworthy that the affinity of the recognition element influences the biosensors specificity,
while the immobilization strategy used has an important impact on the sensitivity [21]. The bioreceptor
can be an enzyme, antibody, nucleic acid, microorganism, cell or a tissue.
Label-Free technology is based on the measurement of the generated signal obtained after the
surface change induced by the analyte. It offers direct information about the interaction of the target
with the sensing element by measuring changes on physical properties such as mass, refractive index,
or electrical resistivity produced by this binding [22]. This change can be monitored by employing
various detection mechanisms: electrical, optical or mechanical. In the first category, we study the
conduction, capacitance or resistance, while in optical devices, changes in light (absorption and
emission) are evaluated, and finally, mechanical sensors are mass and frequency-sensitive devices [23].
In addition to methods based on solid supports, label-free assays can also be performed directly in
solution and are mostly coupled to optical detection.The best example of these assays are sensors
based on the switchable structure of aptamers, aptamer DNAzymes and/or chemical of physical
properties of nanoparticles [24]. Bulbul et al. reported an optical aptasensor based on the alteration of
the catalytic activity of redox active nanoceria and the conformational change of OTA aptamer upon
target binding [25]. The common point of these techniques is the relationship between the measured
signal and the concentration of the bound target. The signal generated from the minimum detectable
change corresponds to the limit of detection (LOD), while the dynamic range is related to the minimum
and the maximum measured levels. Label-Free detection offer new opportunities to food safety field
due to their numerous advantages such as: high sensitivity, simplicity, and possible miniaturization
and portability, which are indispensable for point of care applications [22].

3. Aptamers in Label-Free Biosensing

Increased attention has been recently given to aptamer-based strategies and particularly,
unlabelled assays. This returns to the numerous advantages of these promising recognition
biomolecules over the traditionally used ones. Aptamers are synthetic, single or double stranded,
oligonucleotides selected in vitro by Systematic Evolution of Ligands by EXponantial Enrichment
(SELEX) technology for their ability to recognize a target [26]. In addition to their specificity and
selectivity, the chemical nature and synthesis of aptamers make them cost effective and stable under
extreme conditions. For that, aptamers have rivalled antibodies whose synthesis requires animal
immunization which is expensive and time consuming. Indeed, because of their proteic nature,
antibodies are not stable to certain pH and temperature variations [7,27].
Label-free aptasensing depends on smart interactions between an aptamer, its complementary
strand, target or a signal probe. This unique property of nucleic acids allows the label-free sensing
of a wide variety of targets, including small molecules, while the label-free immunosensing of such
molecules requires more time and efforts [28].

3.1. Label-Free Aptasensing Formats

The formation of aptamer-target complex induces different types of changes where the
aptamer can undorgo configurational or conformational modifications. Depending on this change
label-free aptasensing strategies can be classified into: (1) structure switchable aptamer assays;
(2) aptamer construct assembly/disassembly based assays and (3) target-induced variation in charge
transfer transistance.

3.1.1. Structure Switchable Aptamer Assays

This category of assays exploit the most important advantage of aptamers which consists
in their switchable structure conformation upon the target binding. The ability of aptamers to
form Watson-Crick base pairs, allows the replication of their primary structure onto secondary and
tertiary structures. In contrast to the secondary structure, other base parings are implemented in
Sensors 2016, 16, 2178 5 of 21

2016, 16, 2178 5 of 21

the target induced-three dimensional folding of aptamers, where the paired and unpaired regions
appropriate ligand [29]. Different three dimensional structures contributing in aptamer stabilization
create appropriate structures with which the aptamer can form the ideal fit and bind tightly to its
can be encountered, such as; G-quadruplex, hairpins and stem-loops [30,31].
appropriate ligand [29]. Different three dimensional structures contributing in aptamer stabilization
In aptaswitching detection strategies, aptamers are first folded into a stable three dimensional
can be encountered, such as; G-quadruplex, hairpins and stem-loops [30,31].
structure involving weak and non-covalent bonds; hydrogen bonding, van der Waals interactions
In aptaswitching detection strategies, aptamers are first folded into a stable three dimensional
and hydrophobic effects [32]. Then, the switchable event ocurrs by aptamer destabilization or
structure involving weak and non-covalent bonds; hydrogen bonding, van der Waals interactions and
attachment to a complementary strand. Finally, the conformational change is directly translated into
hydrophobic effects [32]. Then, the switchable event ocurrs by aptamer destabilization or attachment
a measurable signal (Figure 2) [33]. Based on this principle, many aptamer-based assays have been
to a complementary strand. Finally, the conformational change is directly translated into a measurable
reported in the literature in labelled and unlabelled detection formats compatible with various types
signal (Figure 2) [33]. Based on this principle, many aptamer-based assays have been reported in the
of transducers and target compounds [3437]. This flexibility returns to the numerous advantages of
literature in labelled and unlabelled detection formats compatible with various types of transducers
this category of biosensors; the rapid response which can be obtained within few minutes, simple
and target compounds [3437]. This flexibility returns to the numerous advantages of this category of
operation without the need of multiple transduction steps, reusability and reagentless structure
biosensors; the rapid response which can be obtained within few minutes, simple operation without
switching [38,39].
the need of multiple transduction steps, reusability and reagentless structure switching [38,39].

Figure 2. Principle of structure switchable aptamer-assays.

Figure 2. Principle of structure switchable aptamer-assays.

3.1.2. Aptamer Construct

3.1.2. Aptamer Assembly/Disassembly
Construct BasedBased
Assembly/Disassembly Assays
Assays
This category of label-free
This category aptamer-assays
of label-free aptamer-assaysis based on theonmodification
is based the modificationof theofsensors
the sensors
configuration. After formation of the aptamer-target complex, an association
configuration. After formation of the aptamer-target complex, an association or a dissociation or a dissociation of of the
the biosensing construct is induced. The association or assembly leads to the formation
biosensing construct is induced. The association or assembly leads to the formation of a sandwich of a sandwich
structure with awith
structure secondary aptamer.
a secondary On the
aptamer. Onother hand,
the other the dissociation
hand, the dissociation or disassembly
or disassembly results in in the
results
the release of a DNA strand, this strategy is also called target-induced strand displacement
release of a DNA strand, this strategy is also called target-induced strand displacement [40]. Both [40].
Both mechanisms
mechanismscausecausechanges
changesononthe surface
the surface electrode
electrode byby
generating
generating a signal
a signalthat cancan
that be translated
be translated by
by different detection methodologies (Figure 3) [38]. Several strategies have
different detection methodologies (Figure 3) [38]. Several strategies have been developed by been developed by using
using this principle but mostly strand-displacement-based aptasensors [4143]. In
this principle but mostly strand-displacement-based aptasensors [4143]. In the usual design, aDNAthe usual design,
aDNAdouble
doublehelix
helix is
is immobilized
immobilized on on the
the electrode
electrode surface,
surface,in inwhich
whichone oneofofthe strands
the strands contained
contained the
the aptamer sequence. Upon the target binding, the complementary strand is dissociated
aptamer sequence. Upon the target binding, the complementary strand is dissociated generating generating
thus a thus
detectable response.
a detectable By comparison
response. with structure
By comparison switchable
with structure aptamer-assays,
switchable this strategy
aptamer-assays, thiscan
strategy
be generalized, because it because
can be generalized, does notitrequire
does nota prior
requirestudy of the
a prior secondary
study or tertiaryor
of the secondary structure
tertiary of the
structure of
aptamer [44].
the aptamer [44].
 2016,2016,
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2016, 16, 2178 6 of 21

Figure 3. Principle
Figure of target-induced
3. Principle strand
of target-induced displacement
strand aptasensing.
displacement aptasensing.
Figure 3. Principle of target-induced strand displacement aptasensing.
3.1.3. Target-Induced
3.1.3. Target-Induced Variation
Variationin Charge
in Charge Transfer Resistance
Transfer Resistance
3.1.3. Target-Induced Variation in Charge Transfer Resistance
This
Thisaptasensing
aptasensing format
format is the simplest
is the simplest one, because
one, because it isit based
is based on onthethedirect monitoring
direct monitoring of the
of the
This aptasensing
interaction between format
the is theand
aptamer simplest
its one, It
target. because
does it isdepend
not based on on the
a direct monitoring of the
interaction between the aptamer and its target. It does not depend conformational change ofofthe
conformational change
interaction
theaptamer between
aptamerstructure the
structure or aptamer and
or configurational its target.
configurational change of It does
of thenot depend
theaptamer on
aptamerconstruct. a conformational
construct.InInthis kind
this change
kindof of of the
detection
detection
aptamer
scheme, structure
thethebinding or configurational
of the target forms change of
a resistant the aptamer
barrier on on construct.
thethesensing In this
surface kind of
blocking detection
thus thethe
scheme, binding of the target forms a resistant barrier sensing surface blocking thus
scheme,
electron the binding
transfer to theof the target
electrode forms
[45]. a
This resistant
change barrier
in charge on the
resistancesensing surface
is proportionalblocking
to the thus
amountthe
electron transfer to the electrode [45]. This change in charge resistance is proportional to the amount
electron
of of
thethe transfer
target to the
in the electrode
sample. The [45]. This transfer
charge change inresistance
charge resistance
(Rct(R aismanifestation
) ctis) is proportional to ofthe amount
thethe energy
target in the sample. The charge transfer resistance a manifestation of energy
of the target
potential in the with
associated sample.
the The charge
oxidation or transfer resistance
reduction event on (Rctsurface,
the ) is a manifestation
and the energy ofbarrier
the energyof the of
potential associated with the oxidation or reduction event on the surface,and the energy barrier
potential
redox associated with the oxidation or reduction event on the surface,and the energy barrier of
the species reaching
redox species the electrode
reaching due to
the electrode electrostatic
due repulsionor
to electrostatic repulsionor steric hindrance
steric hindrance [19][19]
(Figure
(Figure 4). 4).
the redox
Based species
on this reaching
principle, the
differentelectrode due
aptasensing to electrostatic
schemes repulsionor
have been reported steric hindrance [19]
for the monitoring (Figure 4).
of various of
Based on this principle, different aptasensing schemes have been reported for the monitoring
Based
targets on this
[44,46].
various principle,
targets [44,46].different aptasensing schemes have been reported for the monitoring of
various targets [44,46].

Figure
Figure 4. Principle
4. Principle of target-induced
of target-induced variation
variation in charge
in charge transfer
transfer resistance.
resistance.
Figure 4. Principle of target-induced variation in charge transfer resistance.

3.2.3.2. Aptamer
Aptamer Immobilization
Immobilization Techniques
Techniques
3.2. Aptamer Immobilization Techniques
In In
an an aptasensing
aptasensing scheme,
scheme, thethe biological
biological recognition
recognition cancan
be be analysed
analysed in in solution
solution or or
by by
In an aptasensing scheme, the biological recognition can be analysed in solution or by
immobilizing
immobilizing thethe aptamer
aptamer onto
onto a solid
a solid support.
support. However,
However, thethe immobilization
immobilization technique
technique usedused should
should
immobilizing the aptamer onto a solid support. However, the immobilization technique used should
maintain the affinity and specificity of the aptamer to its target. On the other hand,
maintain the affinity and specificity of the aptamer to its target. On the other hand, it should allow it should allow
maintain the affinity and specificity of the aptamer to its target. On the other hand, it should allow
an easy recovery of the aptamer for multiple regenerations. For that, the aptamer
an easy recovery of the aptamer for multiple regenerations. For that, the aptamer can be physically can be physically
an easy recovery
adsorbed of surface,
the aptamer for multiple regenerations. For that, theoraptamer can be physically
adsorbed on onthethe
surface, covalently
covalently attached
attached with
with functional
functional groups
groups coupled
or coupled to SAMS.
to SAMS. Different
Different
adsorbed on the surface, covalently attached with functional groups or coupled to SAMS. Different
reviews have discussed the surface immobilization methods of aptamers that we summarize in in
reviews have discussed the surface immobilization methods of aptamers that we summarize
reviews have discussed the surface immobilization methods of aptamers that we summarize in
Table
Table 1 [4749].
1 [4749].
Table 1 [4749].
Sensors 2016, 16, 2178 7 of 21

Table 1. Different types of aptamer immobilization techniques for aptasensor applications.

Example of
Technique Principle Advantages Limitations
Bioconjugation
Direct attachment
Electrostatic forces Weak attachment on metals surfaces
Physical adsorption Van der Waals Simple and rapid Random orientation and surfaces coated
interactions of aptamers with hydrophobic
polymers
Interactions between
Wide range Multiple conjugation
the surface functional NHS ester chemistry
Covalent attachment of functional steps Non
groups and aptamers Click chemistry
groups Flexibility specific binding
chemical groups
Amphiphilic
molecules:
hydrophilic and
More suitable with Thiols and alkyne
hydrophobic groups Stability Oriented
SAMs silicon and disulfides on gold
with respective recognition
gold surfaces Alcohols on glass
affinity to the
transducer and
the aptamer

3.3. Detection Methodologies in Label-Free Aptasensing

As mentioned above, label-free aptamer-based assays are adaptable with several transduction
platforms which are sensitive to changes in interfacial properties, we will focus in this review on the
most frequently used techniques; optical, electrochemical and mechanical detection methodologies.

3.3.1. Optical Detection

In an optical biosensing scheme, the interaction of the biocomponent with the target molecule
produces a change in the optical contents of the reflected light. This change is then detected by the
transducer allowing the direct quantification of the target in a given sample [50]. Several types of optical
methods can be used, among which colorimetric and fluorescence ones have gained more attention.

Colorimetric Assays
Colorimetric assays are based on a colour change which occurs in the presence of the target.
The significant advantage of this kind of assay systems is the minimal need for special analytical
instrumentation. In some cases, the results are visible by the naked eye and understandable
without a priori knowledge. This unique property of colorimetry makes this technology a universal
detection method for rapid, low cost and real-time monitoring of a wide variety of analytes [28].
These sensors often involve metal nanoparticles or DNAzymes (aptamers with catalytic activity) for
signal amplification.
Gold nanoparticles (AuNPs) are excellent colorimetric indicators that have been widely associated
to aptasensing schemes. The principle of these techniques is based on the aggregation/dispersion of
AuNPs, determined by the presence or absence of salts, in addition to the key role of DNA in AuNps
stabilization against salt-induced aggregation.The presence of the target in aptasensor system induces
the nanoparticles re-aggregation. This property change from dispersion to aggregation leads to a
colour change from red to blue which is proportional to the amount of analyte in the sample [17].
G-quadruplex-hemin DNAzymes have been also used for signal amplification in several label-free
aptasensing stragies [51,52]. It is well established, that G-rich oligonucleotides can fold intro
G-quadruplex structures stabilized by different metal ions. Certain of these structures are able
to bind to hemin (a peroxidase cofactor) with high specificity and affinity. The formed complex
G-quadruplex-hemin can function as a kind of peroxidase-mimicking DNAzyme. It has been
demonstrated that the catalytic activity of this complex is much higher than that of hemin itself [53].
Sensors 2016, 16, 2178 8 of 21

Based on these finding, an aptasening platform can be constructed by either releasing or blocking the
G-rich DNAs that could form effective hemin-DNAzyme after targeting [28].

Fluorescent Assays
Since standard aptamers and most analytes do not have intrinsic fluorescent properties, fluorescent
biosensors are based on the transduction of the biorecognition event into optical signal by employing
small organic dyes as signal reporters [35]. Most of the label-free fluorescent aptasensors are based
on molecular beacon strategy. A basic molecular beacon is a hairpin-shaped oligonucleotide with
quenched fluorescent marker whose fluorescence is restored by the targeting [54]. In label-free assays,
the principle of molecular beacon strategy is based on the enhancement of the dyes fluorescence
intensity by the aptamer, and its quenching upon the target binding. In aqueous solution, the signal
reporter has a very low fluorescence emission, while the addition of the aptamer induces a remarkable
change in the fluorescent properties. This phenomenon has been explained by Du et al., based on
the fact the DNA can serve as a negative pocket to protect the fluorescent ligands from aggregation
and the emission quenching in aqueous media [28]. Upon formation of the targetaptamer complex,
the target changes the aptamer conformation and blocks the binding site for dyes. The dye is thus
released with an obvious fluorescence decrease. Based on this fluorescence change, the concentration
of the analyte in the sample can be measured. Although its sensitivity and specificity, this strategy is
limited to aptamers exhibiting a target-induced conformational change. For that, a modified format
has appeared as alternative; duplex to complex detection strategy can be performed, in certain cases,
without aptamer conformational change. The aptasensing is based on the transition between aptamer
duplex and ligand-aptamer complex [20].

3.3.2. Electrochemical Detection

In a typical electrochemical aptasensor, the aptamer is immobilized on an electrode surface,
while the recognition event is studied based on electrical current variations. As in label-free fluorescent
assays, redox probes are bound to the aptamer through weak interactions such as electrostatic
adsorption and hydrogen bonds. The target-induced change in the reporters electrochemical
characteristics depends on the amount of the analyte in the sample. Based on the oxidation of aptamer
bases, direct electrochemical detection is also possible. This can be accomplished by monitoring
the oxidation of guanine and adenine which are the most redox-active bases [55]. Electrochemical
Impedance Spectroscopy (EIS) and amperometric methods are widely used in label-free electrochemical
aptasensing strategies.
In a label-free amperometric/voltammetric biosensor, the principle is based on the measurement
of the current generated from the oxidation and reduction of an electroactive species at a fixed or
variable potential. The change in the current corresponds to the concentration of the analyte in the
sample. The amperometric detection can be direct if the target is potentially active, or indirect based
on the redox reaction of an electroactive probe.
EIS allows the sensitive monitoring of changes in conductivity/resistance, or charging capacity
of an electrochemical interface. The modification of a conductive surface, by a recognition
element, induces the formation of an electrical double layer and a change in the resistance of the
electrode-electrolyte interface. This unique property of impedance technology allows the effective
and sensitive probing of affinity binding events [30]. Therefore, based solely on the resistance change,
it is possible to monitor the formation of the complex aptamer-target directly independently to
conformational change or strand displacement. Impedimetric biosensors can be Faradic or non-Faradic;
depending on whether a redox probe is required or not. Non-Faradic impedance is experimentally
simpler, because it does not involve redox processes, thus allowing practical real time monitoring [23].
In general, in both cases, Faradic and non-Faradic impedimetric biosensors, an increase in resistance
is observed after the biorecongnition event. Taking as example, a Faradic strategy where the anionic
[Fe(CN)6 ]3/4 couple is used as redox probe, in this case, the formation of the complex aptamer-target
Sensors 2016, 16, 2178 9 of 21

blocks the redox probe from approaching the electrode surface thus increasing the resistance.
In addition to standard methods, impedimetric label-free aptasensing, based on strand displacement
can be also performed. For that, the aptamer is bound to a complementary strand to form a duplex.
After the target-binding, the aptamer is displaced from the duplex into solution, with a corresponding
decrease in electron transfer resistance [44]. Despite their sensitivity, selectivity and low cost, label-free
impedimetric biosensors still suffer from some limitations such as luck of reproducibility. Theorically,
the electron-transfer rate is selectively modulated by the analyte [56], however, impedance can be
affected by the immobilization technique adopted for the surface functionalization. For example,
the ability of the functional groups to be ionized may have an effect on the charge transfer, influencing
thus the assay reproducibility [19].

3.3.3. Mechanical Detection

Despite the predominance of optical and electrical detection methods in the realm of biosensors,
the increasing knowledge about the mechanical nature of biological mechanisms has led to the
development of promising mechanotransuction processes to detect biological interactions [57].
Piezoelectric materials (ex; quartz) and cantilevers are the most commonly used transducers to monitor
the mass change resulting from the recognition events.In a cantilever-based biosensor, the target
binding is translated into defelection or changes in the resonance frequency of a cantilver which reflects
on the analyte amount in a sample [58], whereas, in quartz-cristal microbalance (QCM) biosensors,
the biocomponent is immobilized on a quartz material which is a stable and sensitive oscillator.
The biochemical reaction decreases the oscillation frequency of the quartz cristal of the microbalance
allowing thus the quantitative detection of the mass deposited on the electrode surface [45,59].

4. Label-Free Aptasensors for Myctotoxin Determination

Mycotoxins are secondary fungal metabolites of low molecular weight that contaminate food
and feedstuffs. They occur more frequently in areas with a hot and humid climate, favorable for
the growth of molds, but they can also be found in temperate zones [60]. Mycotoxins pose serious
health and economic problems, being responsible for mycotoxicosis, at low levels, with symptoms of
intoxication causing substantial effects on animal and human health [5]. Among about 200 known
mycotoxins, ochratoxins, aflatoxins and fumonisins are the most toxic [61]. Aptamers and aptasensors
reported in the literature have been mostly designed for ochratoxin A (OTA), aflatoxins B1, B2 and M1
(AFB1, AFB2 and AFM1), fumonisin B1 (FB1) and zearalenone (ZEA). Table 2 summarizes the different
label-free aptasensors reported in the literature for mycotoxins determination.

Table 2. Label-Free aptasensors for mycotoxin determination in food reported in the literature.

Mycotoxin Detection Assay Principle Linear Range (g/L) LOD (g/L) Sample Ref
OTA Colorimetric HRP mimicking DNAzyme 3.6120 12 Wine [62]
HRP mimicking DNAzyme, Yellow rice, wine,
Colorimetric 0.0040.96 0.004 [63]
Hybridization chain reaction wheat flour
Colorimetric Structure switching 0.0812 0.06 Milk [25]
aptamer Nanoceria
Structure switching aptamer
Fluorescence 0.11 0.02 Wheat [64]
Tb3+ , magnetic sepatation
Structure switching aptamer
fluorescence 1100,000 1 Beer [65]
Pico green dye
Fluorescence SYBR green dye 3.640 3.6 - [66]
Structure switching
Luminescence 260 2 - [67]
aptamer Iridium(III)
Structure switching aptamer
LSPR 0.4400 0.4 Ground corn [68]
Red shift of LSPR band
EIS OTA-induced change in Rct 0.110 0.1 [69]
OTA-induced change Coffee, flour,
EIS 0.0440 0.048 [70]
in Rct [Fe(CN)6 ]3/4 wine
OTA-induced change
EIS 1.25 103 0.5 0.25 103 Beer [71]
in Rct [Fe(CN)6 ]3/4
OTA-induced change
EIS 0.152.5 0.15 Cocoa [72]
in Rct [Fe(CN)6 ]3/4
Structure switching aptamer
EIS
OTA-induced change in Rct 0.12 103 5.5 103 0.12 103 Beer [73]
Structure switching aptamer
CV 0.128.5 0.1 Beer [74]
OTA-induced change in Rct
Sensors 2016, 16, 2178 10 of 21

Table 2. Cont.

Mycotoxin Detection Assay Principle Linear Range (g/L) LOD (g/L) Sample Ref
Structure switching aptamer
EIS 0.0440 0.008 Beer [75]
OTA-induced change in Rct
Structure switching aptamer
EIS 0.00440 0.0056 Wine [22]
OTA-induced change in Rct
Nano-impact Structure switching aptamer
OTA-induced collision 0.0284 0.02 [76]
electrochemistry
frequency changes
AFB2 Structure switching aptamer 0.02510 0.025 Beer
Colorimetric [77]
AuNPs aggregation
AFB1 Colorimetric Structure switching aptamer 0.025100 0.025 [78]
AuNPs aggregation
AFB1 Colorimetric HRP mimicking DNAzyme 0.11.0 104 0.054 Ground corn [79]
AFB1 Chemiluminescence HRP mimicking DNAzyme 0.110 0.11 Corn [80]
OTA and Target-induced strand displacement
Fluorescence 0.0010.05 0.0002 and 0.0003 Rice, corn, wheat [81]
AFB1 DNA-scaffolded silver nanoculsters
Target-induced blocking of
AFM1 CV, SWV 0.0060.06 0.00198 [82]
chargetransfer to the electrode surface
AFM1 EIS Target-induced change in Rct 0.0020.15 0.00115 Milk [83]
AFB1 EIS Target-induced change in Rct 0.12516 0.12 Beer and wine [84]
Target-induced blocking of
AFB1 CV, EIS 0.033.125 0.125 Peanuts [85]
chargetransfer to the electrode surface
Chinese wildrye
hay and infant
AFB1 RT-qPCR Target-induced strand displacement 5 105 5 0.000025 [9]
rice cereal
samples
FB1 EIS target-induced change in Rct 72720 103 1.44 Maize samples [86]
Target-induced change
Microcantilever 33 10040,000 - [87]
in surface stress

4.1. Label-Free Aptasensors for OTA Detection

Because of its widespread occurrence and hazardous effects on animal and human health,
OTA is the most studied mycotoxin in food analysis. Produced by several fungalspecies of the
genera Penicillium and Aspergillus, OTA contaminates a wide variety of food matrices, particularly
cereals, grape products and roasted coffee [88]. Since the selection of OTAs aptamer by a Canadian
group in 2008, various labeled and unlabeled aptasensing strategies have been reported [89]. Despite
the recent advances in label-free biosensing, OTA determination using unlabeled aptasensors is
still a challenge. Our lab has reported a colorimetric assay based on a horseradish peroxidase
(HRP)-mimicking DNAzyme whose advantages have been discussed above. DNAzymes present a
promising alternative to the use of enzymes in aptamer-based biosensors. The label-free aptasensor
was designed using two oligonucleotides. The first comprises OTA-aptamer and DNAzyme sequences
(the principle is shown in Figure 5). The second one consists in a blocking DNA which includes a
partially complementary sequence to a part ofthe aptamer and partially complementary one to a part
of the DNAzyme. The principle of the bioassay is based on the target-induced DNA folding to form
a rigid antiparallel G-quadruplex structure. This conformational change inhibits the hybridization
of the two oligonulceotides thus increasing the catalytic activity of the HRP-mimicking DNAzyme.
The aptasensor was applied on wine samples and the spectrophotometric measurements showed a
linear correlation between DNAzyme activity and OTA concentration in the range of 3.6120 g/L.
However, the LOD of this assay was higher than the maximum tolerated level of OTA in wines
established by the European Commission (2 g/L) [62]. Wang et al. combined the advantages
of G-quadruplex DNAzymes and hybridization chain reaction technology (HCR). HCR is a signal
amplification technique, based on the chain reaction of recognition and hybridization events between
two DNA hairpin molecules. The aptasensor was designed by using two hairpin oligonucleotides, the
aptamer is localized in the 3-end of the first molecule while the DNAzyme is in the central part of the
second hairpin. The target binding induces the opening of the hairpin structure and the hybridization
chain reaction releasing thus many DNAzymes. This HCR generates enhanced colorimetric signals,
which is correlated to the OTA concentration in the sample. The reported aptasensor showed a high
sensitivity (LOD of 0.004 g/L) [63].
Recently, Bulbul et al. reported a colorimetric method based on the conformational transition
state of the aptamer on nanoceria, combined with the redox properties of these particles. Changes
in the redox properties at the nanoceria surface upon binding of the ssDNA and its target analyte
enables rapid and highly sensitive measurement of OTA. By binding different aptamer concentrations
with a fixed OTA concentration, the association constant of aptamer-OTA conjugate on nanoceria
Sensors 2016, 16, 2178 11 of 21

was
2016, calclulated
16, 2178 as 0.120 nmol1 . In addition, the authors have shown that the optical properties
11 of of
21
nanoceria are conserved and enhanced after 2 days [25].

Figure 5. Principle
Figure 5. Principle of
of aa colorimetric
colorimetric aptasensor
aptasensor based
based on
on HRP-mimicking
HRP-mimicking DNAzyme.
DNAzyme. Incubation
Incubation of
of
the
the aptasensor
aptasensor with
with OTA
OTA reduces
reduces the
the affinity
affinity between
between the
the first
firstoligonucleotide
oligonucleotide (aptamer+DNAzyme)
(aptamer+DNAzyme)
and
and the
the second
second one
one which
which is is the
the blocker
blocker increasing
increasing thus
thus the
the HRP
HRP activity
activity [53].
[53].

Simple label-free fluorescent aptasensors have been also reported for OTA analysis to overcome
fluorphore-labeled aptamers limitations. limitations. TheseThese strategies
strategies are
are mostly
mostly basedbased on an enhancement in
the fluorescence intensity of a dye by by intercalation
intercalation with
with an an aptamer.
aptamer. First, Zhang et al. al. reported a
3+)3+
fluorescent aptasensor
aptasensor based based on onthetheability
abilityofofssDNA
ssDNAtotoenhance
enhancethe theemission
emissionofof terbium
terbium ion(Tb
ion(Tb in)
solution.
in solution. TheTheaptasensor
aptasensor was
was constructed
constructed bybyimmobilizing
immobilizingOTA OTAaptamer
aptameron onmagnetic
magneticbeads beads(MBs).
(MBs).
In absence of OTA, the aptamer hybridized with two ssDNA probes present in the solution, blocking
thus the Tb3+ 3+ emission. Upon addition of OTA, the aptamer structure switched to G-quadruplex and
emission. Upon addition of OTA, the aptamer structure
released the thessDNA
ssDNA probes
probes resulting
resulting in an enhancement
in an enhancement in Tb3+ fluorescence
in Tb3+ fluorescence in solution
in solution proportional
proportional
to the amount to the amount ofa OTAwith
of OTAwith a limit of as
limit of detection detection
low as as 0.02lowg/Las 0.02 g/LIn[64].
[64]. to Tb3+
In contrast
contrast to,
Tb 3+, Pico Green reagent (an asymmetric cyanine dye) fluoresces upon binding to dsDNA, while
Pico Green reagent (an asymmetric cyanine dye) fluoresces upon binding to dsDNA, while ssDNA does
ssDNA
not changedoesits not change its
fluorescence fluorescence
intensity. Based on intensity. Based Lv
this principle, onetthis principle, Lv
al. developed et al. developed
a fluorescent label-freea
fluorescent for
aptasensor label-free aptasensor forinOTA
OTA determination beerdetermination
samples. The in beer samples.
detection was basedThe on detection was based
a competition for
on a competition
aptamer for aptamer between
between aptamer/OTA complexaptamer/OTA
and aptamer/cDNA complex and aptamer/cDNA
duplex, where, the quantity duplex,
of OTAwhere,
was
the quantity
inversely of OTA was
proportional inversely
to the proportional tointhe
fluorescenceintensity thefluorescenceintensity
wide linear range of in ( 1 the wide linear
to 100,000 g/L) range
[65].
of ( 1 to 100,000McKeague
Furthermore, g/L) [65].etFurthermore,
al. used another McKeague et al. used
fluorescent dye another
(SYBR green)fluorescent
whose dye (SYBR green)
fluorescence is
whose fluorescence
enhanced upon binding is enhanced
to dsDNA. upon bindingconcentrations
Increasing to dsDNA. Increasing
of the OTA concentrations of the OTA
caused a displacement of
causedgreen
SYBR a displacement
from the aptamer,of SYBR and green from the aptamer, andloss
a concentration-dependent a concentration-dependent
of emitted fluorescence. loss In thisof
emitted fluorescence. In this work the authors selected a new specific
work the authors selected a new specific aptamer for OTA that showed similar characteristics to aptamer for OTA that showed
similar
the characteristics
original one. However, to the the
original
aptamer one. shows
However, the aptamer
modest affinity to shows
OTB modest affinity response
and negligible to OTB and to
negligible
wafarin response
[66]. In additionto wafarin [66]. In
to fluorescent addition
probes, to fluorescent
luminescent probes probes,
were also luminescent
investigatedprobes were
in label-free
also investigated
aptasensing of OTA. in Lulabel-free aptasensing
et al. used luminescent of metal
OTA. complexes
Lu et al. used luminescent
(octahedral metalselective
Iridium(III)) complexes for
(octahedral Iridium(III))
G-quadruplex structures selective
to constructfor aG-quadruplex structuresTwo
switch-on aptasensor. to construct a switch-on
oligonucleotides haveaptasensor.
been used;
Twofirst
the oligonucleotides
comprising OTA have been used;
aptamer, the firstwith
hybridized comprising
a partiallyOTA
cDNA aptamer,
strand.hybridized
OTA binding withinduced
a partially
the
cDNA strand.
disassembly of OTA binding
the duplex, induced
allowing thethe disassembly
aptamer to fold of thea quadruplex
into duplex, allowing structure the and
aptamer to fold
adecrease of
into a quadruplex
luminescence. structuresuggested
The authors and adecrease of duplex
that this luminescence.
to complexTheapproach
authors suggested
is advantageous that this duplex
compared
to acomplex
randomapproach is advantageous
coil-to-quadruplex strategies,compared
becausetoofa the
random coil-to-quadruplex
resistance of initial duplex strategies,
substratebecause
against
of the resistance of initial duplex substrate against the presence of interfering ionswhich may induce
aptamer folding even without the addition of OTA [67].
Despite the sensitivity of the mentioned optical aptasensors, their reusability was not
demonstrated. Park et al. developed a regenerable label-free localized surface plasmon resonance
Sensors 2016, 16, 2178 12 of 21

the presence of interfering ionswhich may induce aptamer folding even without the addition of
OTA [67].
Despite the sensitivity of the mentioned optical aptasensors, their reusability was not
demonstrated. Park et al. developed a regenerable label-free localized surface plasmon resonance
(LSPR) aptasensor based on a structure switching aptamer and gold nanorods. The detection of OTA
was achieved by monitoring the change in the magnitude of the LSPR wavelength which depended on
the location of the analyte relative to the surface of the nanoparticle and the degree of alteration of the
refractive index. Importantly, the biosensor was regenerated by heating in methanol at 70 C [68].
EIS is the most employed method for label-free aptasensing of OTA in food samples. This is due
to the high sensitivity, low cost, fast response time and simple equipment. Aptamer immobilization
and the oriented organization of biomolecules on the sensor play a key role in the performance
of a biosensor. For that, several immobilization strategies have been reported in the literature for
impedimetric label-free aptasensing. In 2011, Prabhakar et al. described the first impedimetric
label-free aptasensor for OTA detection. In this report, OTA aptamer was covalently immobilized onto
mixed LangmuirBlodgett (polyanilinestearic acid) film deposited onto indium tin-oxide coated glass
plates. Then, the change in the magnitude of transfer resistancedue to OTA binding observed at the
sensor surface is utilized for sensitive detection of OTA. The aptasensor is regenerable by disrupting
aptamer-OTA complex in 50mMNaOH for 2min [69]. Despite its sensitivity (LOD of 0.1 g/L),
the fabrication procedure of this sensor required complicated immobilization steps as compared to
the simple chemisorption reported by Castillo et al. In this work, the thiolated aptamer with different
configurations was chemisorbed on the surface of a gold electrode. OTA determination was based
on the monitoring of charge transfer resistance which increased with increasing OTA concentration
in the sample. A redox probe [Fe(CN)6 ]3/4 , was used to amplify the detection of the interaction
aptamer-OTA. The sensor was validated with coffee, floor and wine samples with high sensitivity
(LOD of 0.048 g/L) [70]. Furthermore, we designed in our lab different label-free aptasensors for
OTA determination by using innovative immobilization chemistries. In the first one, azido-aptamer
was immobilized onto an electrografted binary film by click chemistry. After modification of ascreen
printed carbon electrode (SPCE) surface with a layer of active ethynyl groups, the latter reacted
efficiently with aptamer bearing an azide function in the presence of copper (I) catalyst. The increase
in electron-transfer resistance values due to the specific aptamerOTA interaction was proportional
to the concentration of OTA where the LOD achived 0.00025 g/L [71]. We have demonstrated that
the immobilization via click chemistry improved the aptamer binding capacity and magnified the
response signal of the aptasensor. For that, the same strategy was exploited in another report for
on-site monitoring of OTA in cocoa beans and cocoa powder [72]. Later on, we proposed another
immobilization strategy for label-free electrochemical OTA aptasensing. The sensor was based on two
piece macromolecules; an aminomodified aptamer covalently attached to the carboxy end of a PEG
(polyethylene glycol) spacer immobilized on a SPCE surface. In this work, the LOD was two fold
lower than that obtained by using click chemistry (LOD= 0.00012). These macromolecules formed long
tunnels on SPCE surface, while aptamer acted as gate of the tunnels. The principle of this aptasensor is
based on targeted induced conformational changes, OTA binding closed aptamer gates, decreasing the
electrochemical signal [73]. In addition to its high sensitivity, the reported aptasensor required less
time and simpler operation as compared to the previously described aptasensors. The same principle
was used by employing hexamethyldiamine instead of PEG spacer, while electrochemical detection of
OTA was performed by cyclic voltammetry (CV) [22].
Nanomaterials have been also exploited in the development of impedimetric aptasensors because
they present a promising tool for electrode surface modification. This is due to their numerous
advantages such as good conductivity and large surface area. Evtugyn et al. attached a thiolated
aptamer to AuNPs stabilized by a hyperbranched polymer; dendrimeric hydrophilic Boltorn H30 .
The formation of OTA-aptamer complex induced the conformational switch of the aptamer from
linear to guanine quadruplex leading to the consolidation of the surface layer and an increase of the
Sensors 2016, 16, 2178 13 of 21

charge transfer resistance [75]. Recently, iridium oxide (IrO2 ) NPs have been also used for aptamer
immobilization; SPCE surface was modified with an electropolymerized film of polythionine followed
by the assembly of IrO2 NPs. The amino-modified aptamer was subsequently exchanged with the
citrate ionssurrounding IrO2 NPs via electrostatic interactions with the same surface. It is well
established that electropolymerization improves the conductivity and provides a stable redox-active
coatings on the electrode surface. The reported method exhibited the low LOD of 0.0056 g/L and
a linear range of (0.00440 g/L) [76]. The use of silver nanoparticles has been also reported for
quantitative investigation of OTA using nano-impact electrochemistry. The principle of this method is
based on the target-induced changes in collision frequency. These changes are assigned to the surface
coverage of nanoparticles by the aptamer and the conformational change of aptamer which affected
the electron transfer between the electrode and silver nanoparticles [76].

4.2. Label-Free Aptasensors for Aflatoxins Detection

Aflatoxins are toxic metabolites produced by filamentous fungi like Aspergillus flavus and
Aspergillus parasiticus. They are present in agricultural products and animal feeds, including tree
nuts, peanuts, peanut butter, figs and corn. They are responsible forserious human health disorders:
hepatocellular carcinoma, aflatoxicosis, Reyes syndromeand chronic hepatitis. Several types of
aflatoxins are known and classified into six subtypes: aflatoxin B1, B2, G1, G2, M1 and M2, aflatoxin
B1 is the most predominant and toxic class [78]. AFB1 aptamer was first selected and patented
by Neoventures Biotechnology Inc. (London, ON, Canada) [90]. Then, Wangs group identified
specific aptamers for AFB1 and B2 [91], while AFM1 aptamer has been selected by Malhotra et al. [92].
After selection, these aptamers have been used, in many reports, as biorecognition elements in optical
and electrochemical label-free aptasensing strategies.
Based on salt-induced AuNPs aggregation phenomenon, Luan et al. described two colorimetric
label-free aptasensors for AFB1 and AFB2 detection. In the absence of the target, the nanoparticles
were stabilized and dispersed by the aptamer leaving the solution red under high NaCl conditions.
After that, the target-induced conformational change exposes the AuNPs to NaCl-induced aggregation
leading to a colour change. The linear dynamic range and detection sensitivity were found to be
0.025100 g/L and 0.025 g/L of AFB1, respectively [77,78]. Seok et al. reported another colorimetric
assay based on AFB1-induced DNA structural changes and peroxidase mimicking DNAzyme. For that,
the aptamer was combined with split halves of hemin-binding DNAzymes. AFB1 binding induced a
structural deformation of the aptamer-DNAzyme complex, which caused splitting of the DNAzyme
halves thus decreasing peroxidase mimicking activity and the colour signal in the wide linear range of
0.11.0 104 g/L [79]. Based on the same principle of HRP mimicking DNAzymes, a competitive
chemiluminescent aptasensor has been developed for AFB1 detection in corn samples. In this work,
the AFB1 aptamer linked with a dual HRP-DNAzyme produced sufficient chemiluminescence(CL)
values when binding to AFB1-ovalbumin (OVA) used as a coating antigen. The assay was based
on the monitoring of the CL produced from the interaction between the aptamer/HRP-DNAzymes
and luminal. Analytical performances of the sensor have been validated on corn samples where the
LOD attained 0.11 g/L and the extraction recoveries averaged from 60.4% to 105.5% [80]. Recently,
a label-free fluorescent aptasensor was developed for the simultaneous detection of OTA and AFB1 in
cereals. The corresponding aptamers, hybridized with ssDNA signal probes, have been immobilized
on MBs. After the target-induced strand displacement followed by magnetic separation, the released
probes present in the supernatant acted as the corresponding scaffolds to synthesize silver nanoclusters
with different photoluminescence emission bands. The authors have noted an increase in fluorescence
intensity after adding Zn(II)-ion into the system. In addition to its sensitivity (LOD = 0.0002 g/L),
the reported method allowed the discrimination between the two mycotoxins in food samples [81].
Owing to their low cost, simplicity and high sensitivity, electrochemical aptasensors have been
also applied successfully to aflatoxins. Nguyen et al.employed CV and square wave voltammetry to
monitor the biomolecular interaction aptamer-AFM1. For that, the aptamer has been immobilized on
Sensors 2016, 16, 2178 14 of 21

Fe3 O4 incorporated polyaniline film polymerized on interdigitated electrode (IDE). AFM1 binding
strongly influenced the switching rate of polyaniline film and decreased the current, after blocking the
charge transfer to the electrode surface, which is inversely proportional to the analyte concentration.
The principle of the signal-on detection, shownin Figure 6, is based on the competitive reactions
between AFM1-aptamer complex and free aptamer present in solution occurring by virtue of
equilibrium displacement. The treatment of the electrode with an aptamer-rich solution
2016, 16, 2178
induced
14 of 21
the dissociation of aptamer-AFM1 complexes, where the released AFM1 left the electrode surface to the
occurring solution.
aptamer-rich by virtueThe of described
equilibrium displacement.
biosensor The high
has shown treatment of the(0.00198
sensitivity electrode with excellent
g/L), an
aptamer-rich solution induced the dissociation of aptamer-AFM1 complexes,
stability and reproducibility. However, the applicability of the method for AFM1 determination in where the released
AFM1 leftwas
real samples the electrode surface to the
not demonstrated [82].aptamer-rich solution.
Recently, our The described
research biosensor
group reported anhas shown
impedimetric
high sensitivity (0.00198 g/L), excellent stability and reproducibility. However, the applicability of
aptasensor for AFM1 detection in milk samples. In this work, the aptamer, modified with
the method for AFM1 determination in real samples was not demonstrated [82]. Recently, our
hexaethyleneglycol, was covalently immobilized on a SPCE surface activated with diazonium
research group reported an impedimetric aptasensor for AFM1 detection in milk samples. In this
salts.work,
The the method wasmodified
aptamer, successfully applied toreal milk samples
with hexaethyleneglycol,was covalentlywith a good correlation
immobilized on a SPCE with
a conventional
surface activatedimmunoassay
with diazonium[83].salts.
UsingThe the same
method wasimmobilization strategy,
successfully applied toreal wemilkdescribed
samples an
impedimetric aptasensor for AFB1 by comparing two different aptamers. Both
with a good correlation with a conventional immunoassay [83]. Using the same immobilization aptasensors showed
high strategy,
sensitivity, reproducibility,
we described fast response
an impedimetric aptasensorandforgood
AFB1 selectivity.
by comparingThe biosensors
two different have been
aptamers.
testedBoth
on aptasensors showed high
alcoholic beverages sensitivity,
with promising reproducibility, fast response(92%
recovery percentages and good
to 102%)selectivity.
[84]. The
Another
biosensors
original have been tested
immobilization platformon alcoholic
was adoptedbeverages with promising
by Castillo recovery
et al. to attachpercentages
AFB1 aptamer (92% to for the
102%) [84]. Another original immobilization platform was adopted by Castillo
development of an electrochemical aptasensor. The amino-modified aptamer was immobilized on et al. to attach AFB1
aptamer for the development of an electrochemical aptasensor. The amino-modified aptamer was
gold electrode covered with cystamine-poly(amidoamine) dendrimers layer. The thickness of this
immobilized on gold electrode covered with cystamine-poly(amidoamine) dendrimers layer. The
layer decreased after AFB1 binding, this was explained by target-induced conformational change
thickness of this layer decreased after AFB1 binding, this was explained by target-induced
of aptamer. The electrochemical
conformational change of aptamer.detection revealed thatdetection
The electrochemical the current peaksthat
revealed decreased
the currentby peaks
increasing
aflatoxin
decreased by increasing aflatoxin concentration in the dynamic linear range (0.033.125 g/L).the
concentration in the dynamic linear range (0.033.125 g/L). The reusability of The sensor
was demonstrated
reusability of the bysensor
usingwas0.2demonstrated
M glycine-HCl [85]. 0.2 M glycine-HCl [85].
by using

Figure 6. Principle of label-free aptasensor of AFM1 based on Fe3O4 /polyaniline film polymerization
Figure 6. Principle of label-free aptasensor of AFM1 based on Fe3 O4 /polyaniline film polymerization
on interdigitated electrode. (A) Functionalization of the surface with AFM1 aptamer; (B) Signal-off
on interdigitated electrode. (A) Functionalization of the surface with AFM1 aptamer; (B) Signal-off
experiment performed after target binding; (C) Signal-on detection achieved by treating the
experiment performed after target binding; (C) Signal-on detection achieved by treating the biosensor
biosensor with aptamer rich solution which results in the displacement of some AFM1 that leave the
with electrode
aptamer torich
go solution which results
into the solution in the displacement
where aptamer concentrationof some AFM1
is higher [82]. that leave the electrode to
go into the solution where aptamer concentration is higher [82].
Finally, real-time quantitative polymerase chain reaction (RT-qPCR) can be also used as a
detection method of
Finally, real-time biomolecular
quantitative interactions.
polymerase Guo reaction
chain et al. reported a simple
(RT-qPCR) canand
be low
alsocost
usedaptasensor
as a detection
based on target-induced strand displacement. In this detection strategy, AFB1 aptamer was
method of biomolecular interactions. Guo et al. reported a simple and low cost aptasensor based on
hybridized with its complementary strand which acted as a signal generator for PCR amplification.
target-induced strand displacement. In this detection strategy, AFB1 aptamer was hybridized with its
The aptasensing was achieved by monitoring the amplification signal which was related to AFB1
complementary strand
concentration. whichtoacted
In addition as a signal
its simplicity generator
(the whole sensingforprocedure
PCR amplification. The aptasensing
was accomplished in a single was
achieved by monitoring the amplification signal which was related to AFB1 concentration.
PCR tube), the reported method israpid, low cost and highly sensitive (LOD = 0.000025 g/L) [9]. In addition
to its simplicity (the whole sensing procedure was accomplished in a single PCR tube), the reported
method4.3. Label-Free Aptasensors
israpid, low cost and forhighly
Other Mycotoxins
sensitive (LOD = 0.000025 g/L) [9].
Two aptamers have been selected by different research groups for the carcinogenic mycotoxin
fumonisin B1 [93,94]. This mycotoxin, produced by Fusarium moniliforme, occurs mainly in maize and
in processed maize products and animal feeds. After selection and characterization of FB1 aptamer,
Wangs group reported an impedimetric aptasensor for FB1 detection. The thiolated aptamer was
Sensors 2016, 16, 2178 15 of 21

4.3. Label-Free Aptasensors for Other Mycotoxins

Two aptamers have been selected by different research groups for the carcinogenic mycotoxin
fumonisin B1 [93,94]. This mycotoxin, produced by Fusarium moniliforme, occurs mainly in maize
and 16,
2016, in2178
processed maize products and animal feeds. After selection and characterization 15 of ofFB1
21
aptamer, Wangs group reported an impedimetric aptasensor for FB1 detection. The thiolated aptamer
anchored on AuNPs
was anchored directly
on AuNPs electrodeposited
directly on a glassy
electrodeposited carbon
on a glassy electrode
carbon (GCE).
electrode AuNPs
(GCE). have been
AuNPs have
used
been for
usedtheir electrical
for their conductivity
electrical andand
conductivity easeease of of
self-assembly
self-assembly through
throughathiol
athiolgroup.
group.AfterAfterthethe
incubation
incubation of aptasensor with
of the aptasensor with FB1,
FB1,the
theelectron
electrontransfer
transferbetween
between thethe [Fe(CN)
[Fe(CN) ] 36]3/4
/4 electrolyte
electrolyte
6
solution
solution and
and the
the electrodewas
electrodewas significantly
significantly inhibited,
inhibited, resulting
resultinginin aa corresponding
corresponding increase
increase in in the
the
resistance
resistancemonitored
monitored by byEIS
EIS(Figure
(Figure7).
7).Finally,
Finally, the
the applicability
applicability ofof the
the method
methodwaswasproved
provedon onmaize
maize
samples
sampleswith
withsatisfactory
satisfactory results:
results: the
the extraction
extraction recoveries
recoveries ranged
ranged from
from 91%
91% to
to 105%
105% [86].
Label-free
Label-freemass
masssensitive
sensitiveaptasensors
aptasensorshave
havebeenbeenalso
alsoinvestigated
investigatedin inmyctotoxin
myctotoxindetermination.
determination.
Chen al.functionalizedthe
Chen et al.functionalized thesensing
sensing cantilevers
cantilevers in array
in the the array withassembled
with self self assembled
monolayers monolayers
(SAMs)
(SAMs) of thiolated
of thiolated FB1 aptamer.
FB1 aptamer. Aiming to Aiming to avoid interferences
avoid interferences in the environment,
in the environment, reference
reference cantilevers
cantilevers
were modified werewith
modified with 6-mercapto-1-hexanol
6-mercapto-1-hexanol SAMs. ThenSAMs. Then , concentration
, the analyte the analyte concentration
was proportional was
proportional to the
to the diffential diffentialamplitude
deflection deflection between
amplitude between
sensing andsensing andcantilevers
reference reference cantilevers
with a lowwith LOD a
low LOD (33
(33 g/L) g/L) [87].
[87].

Figure
Figure 7.7. Principle
Principleof of impedimetric
impedimetric aptasensor
aptasensor fordetection.
for FB1 FB1 detection. Niquist
Niquist plots of theplots of the
impedimetric
impedimetric
aptasensor beforeaptasensor before
(A) and after (A) andwith
incubation after incubation
FB1 (B) [86]. with FB1 (B) [86].

The same research group selected a specific aptamer for the mycotoxinzearalenone,
The same research group selected a specific aptamer for the mycotoxinzearalenone, anon-steroidal
anon-steroidal estrogenicmycotoxin produced by Fusarium graminearum on maizeand barley [95].
estrogenicmycotoxin produced by Fusarium graminearum on maizeand barley [95]. However,
However, to our knowledge there are no reports on label-free aptamer-based determinationfor
to our knowledge there are no reports on label-free aptamer-based determinationfor zearalenone.
zearalenone.
5. Limitations and Challenges of Aptasensors
5. Limitations and Challenges of Aptasensors
Despite the numerous advantages of aptamers as bioreceptors and aptasensors as promising
Despite
analytical the numerous
devices, advantages
they still of some
suffer from aptamers as bioreceptors
limitations and moreandefforts
aptasensors as promising
are needed to allow
analytical
their successful applicability in complex samples. As they are nucleic acid biopolymers, their
devices, they still suffer from some limitations and more efforts are needed to allow thein
successful applicability in complex samples. As they are nucleic acid biopolymers, thein situ
application of aptamers is crucially limited by their inherent physicochemical characteristics. For
overcoming that,various analytical parameters should be considered and predetermined during the
selection procedure of aptamers. The target should be bound to the random oligonucleotide library
in a selection environment with certain experimental conditions such as:temperature, pH, ionic
Sensors 2016, 16, 2178 16 of 21

situ application of aptamers is crucially limited by their inherent physicochemical characteristics.

For overcoming that, various analytical parameters should be considered and predetermined during
the selection procedure of aptamers. The target should be bound to the random oligonucleotide
library in a selection environment with certain experimental conditions such as:temperature, pH, ionic
strength and buffer components. These conditions are studied according to the sensing environment in
which the target will be assessed. They contribute in the specific selection stringency improving thus
the aptamers affinity and function [96,97].Besides these characteristics, selectivity plays a key role
in the performance of an analytical device. Selectivity of aptamers can be enhanced by introducing a
negative SELEX step. In this counter selection, the random library is incubated with analogue targets,
excluding thus, the candidates exhibiting an affinity to these targets [98].
The possibility to realize aptamer selection under in vitro conditions opens the door for a wide
range of post-SELEX modifications. It has been shown that post-SELEX modification can result in
further enhancement of binding affinity and specificity, as well as other desired properties [99]. Indeed,
post-SELEX modifications allow the bioconjugation of aptamers to transducers, labeling biomolecules
and signal amplificators in order to enhance the aptasensors sensitivity.
Finally, although the promising advantages of aptamers over antibodies, immunoassays still
substantially dominate the market. This is due to the limited number of aptamers available, whether
for biomedical or food safety applications.

6. Conclusions and Future Prospects

Observing molecular binding events, especially for small size molecules, requires the design of
methods that are sensitive to a very small amount of change in the interface of the sensor, to generate
a detectable signal. To date, there are no general labelled sensing platforms that can be applied
for very sensitive and selective detection of small size molecules for real time applications. Major
limitations include, but are not limited to time consuming labelling steps, non-native signal interference,
non-suitability for decentralized analysis, need of highly sophisticated and costly equipment, and the
requirement forskilled persons to operate these equipment.
The development of label-free methodologies, however, has overcome these limitations and a
success level is achieved in this direction to certain extent. Label-free methodologies output a signal that
is directly related to the interaction of target analyte with the biomolecule, contrary to label methods
where the signal is based on the label-attached molecules, and subsequently issues of false positive
and labelling process can be avoided. Moreover, it is expected that label-free sensing methodologies
are more reproducible as compared to those based on the labelling of molecules. The integration
of aptamers as bioreceptor elements in the design of label-free biosensors has further improved the
analytical performance of the assays. The long term stability and in vitro production of the aptamer
molecule along with label-free detection methodologies makes these assays very cost-effective and
highly suitable for field analysis. The phenomenon of aptamer conformational changes upon target
analyte binding offers numerous advantages in the fabrication of label-free methods, as antibody-based
label-free methods are based on the mass changes and label-free immunosensors are limited to a few
assay formats. While aptamers offer additional formats such as those based on the signal on and
signal off detection approaches. In this context, nanomaterials have been successfully integrated with
aptamer to design label-free assays in solution. The electrostatic attachment or the simple adsoption of
aptamer alters the optical properties of the nanomaterials, which are restored in the presence of target
analyte. Similary alteration in the redox properties of nanoparticles upon aptamer conguation, and
subsequent incubation of analyte have been used for the designed optical aptasensors. These assays
permits very fast analysis of target analytes and have comparable analytical performance with those
based on the label methodologies.
In this review paper, we have further shown that how these label-free aptasensor methods are
successfully employed for the detection of the small size molecules of mycotoxins. Although, a great
level of success has been achieved in the domain ofaptamer-based label-free detection of mycotoxins,
Sensors 2016, 16, 2178 17 of 21

however, there are still some gaps to be filled by researchers. For example, all types of mycotoxins
are toxic to a certain level and their monitoring is equally important. However, most of the label-free
aptasensors reported in the literature are employed for the detection of ochratoxin A and aflatoxin
detection. Aptamers are not selected against all type of mycotoxins, future research may focus in this
direction to develop new aptamer sequences for these mycotoxins. Similarly, most of these aptamer
label-free assays are based on the electrochemical transducer platform or performed in solution. Filter
paper has emerged as a very cheap and attractive optical transducer platform foron-site analysis of
various contaminants which can be exploited in the design of label-free aptasensors for the detection
of mycotoxins.

Conflicts of Interest: The authors declare no conflict of interest.

References
1. Mejri Omrani, N.; Hayat, A.; Korri-Youssoufi, H.; Marty, J.L. Electrochemical biosensors for food security:
Mycotoxins detection. In Biosensors for Security and Bioterrorism Applications; Nikolelis, D.P., Nikoleli, G.-P.,
Eds.; Springer International Publishing: Cham, Switzerland, 2016; pp. 469490.
2. Bazin, I.; Tria, S.A.; Hayat, A.; Marty, J.L. New biorecognition molecules in biosensors for the detection of
toxins. Biosens. Bioelectron. 2016, 87, 285298. [CrossRef] [PubMed]
3. Lehninger, A.L. Biochemistry, 2nd ed.; Worth Publishers: New York, NY, USA, 1977.
4. Byfield, M.P.; Abuknesha, R.A. Biochemical aspects of biosensors. Biosens. Bioelectron. 1994, 9, 373400.
[CrossRef]
5. Catanante, G.; Rhouati, A.; Hayat, A.; Marty, J.L. An overview of recent electrochemical immunosensing
strategies for mycotoxins detection. Electroanalysis 2016, 28, 17501763. [CrossRef]
6. Hammers, C.M.; Stanley, J.R. Antibody phage display: Technique and applications. J. Investig. Dermatol.
2014, 134, 15. [CrossRef] [PubMed]
7. Jayasena, S.D. Aptamers: An emerging class of molecules that rival antibodies in diagnostics. Clin. Chem.
1999, 45, 16281650. [PubMed]
8. Hayat, A.; Paniel, N.; Rhouati, A.; Marty, J.-L.; Barthelmebs, L. Recent advances in ochratoxin a-producing
fungi detection based on pcr methods and ochratoxin a analysis in food matrices. Food Control 2012, 26,
401415. [CrossRef]
9. Guo, X.; Wen, F.; Zheng, N.; Luo, Q.; Wang, H.; Wang, H.; Li, S.; Wang, J. Development of an ultrasensitive
aptasensor for the detection of aflatoxin B 1. Biosens. Bioelectron. 2014, 56, 340344. [CrossRef] [PubMed]
10. Yang, L.T.; Fung, C.W.; Cho, E.J.; Ellington, A.D. Real-Time rolling circle amplification for protein detection.
Anal. Chem. 2007, 79, 33203329. [CrossRef] [PubMed]
11. Weizmann, Y.; Beissenhirtz, M.K.; Cheglakov, Z.; Nowarski, R.; Kotler, M.; Willner, I. A virus spotlighted by
an autonomous DNA machine. Angew. Chem. Int. Ed. Engl. 2006, 45, 73847388. [CrossRef] [PubMed]
12. Patolsky, F.; Katz, E.; Willner, I. Amplified DNA detection by electrogenerated biochemiluminescence and
by the catalyzed precipitation of an insoluble product on electrodes in the presence of the doxorubicin
intercalator. Angew. Chem. Int. Ed. Engl. 2002, 41, 33983402. [CrossRef]
13. Deng, C.Y.; Chen, J.H.; Nie, Z.; Wang, M.D.; Chu, X.C.; Chen, X.L.; Xiao, X.L.; Lei, C.Y.; Yao, S.Z. Impedimetric
aptasensor with femtomolar sensitivity based on the enlargement of surface-charged gold nanoparticles.
Anal. Chem. 2009, 81, 739745. [CrossRef] [PubMed]
14. Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. Aptamer-Functionalized Au nanoparticles for the amplified
optical detection of thrombin. J. Am. Chem. Soc. 2004, 126, 1176811769. [CrossRef] [PubMed]
15. Schmitteckert, E.M.; Schlicht, H.-J. Detection of the human hepatitis b virus x-protein in transgenic mice
after radioactive labelling at a newly introduced phosphorylation site. J. Gen. Virol. 1999, 80, 25012509.
[CrossRef] [PubMed]
16. Duan, N.; Wu, S.-J.; Wang, Z.-P. An aptamer-based fluorescence assay for ochratoxin A. Chin. J. Anal. Chem.
2011, 39, 300304. [CrossRef]
17. Yang, C.; Wang, Y.; Marty, J.L.; Yang, X. Aptamer-Based colorimetric biosensing of ochratoxin a using
unmodified gold nanoparticles indicator. Biosens. Bioelectron. 2011, 26, 27242727. [CrossRef] [PubMed]
Sensors 2016, 16, 2178 18 of 21

18. Rhouati, A.; Hayat, A.; Hernandez, D.B.; Meraihi, Z.; Munoz, R.; Marty, J.-L. Development of an automated
flow-based electrochemical aptasensor for on-line detection of ochratoxin A. Sens. Actuators B Chem. 2013,
176, 11601166. [CrossRef]
19. Daniels, J.S.; Pourmand, N. Label-Free impedance biosensors: Opportunities and challenges. Electroanalysis
2007, 19, 12391257. [CrossRef] [PubMed]
20. Li, B.; Dong, S.; Wang, E. Homogeneous analysis: Label-free and substrate-free aptasensors. Chem.An
Asian J. 2010, 5, 12621272. [CrossRef] [PubMed]
21. Hunt, H.K.; Armani, A.M. Label-Free biological and chemical sensors. Nanoscale 2010, 2, 15441559.
[CrossRef] [PubMed]
22. Rivas, L.; Mayorga-Martinez, C.C.; Quesada-Gonzlez, D.; Zamora-Glvez, A.; de la Escosura-Muiz, A.;
Merkoi, A. Label-Free impedimetric aptasensor for ochratoxin-a detection using iridium oxide nanoparticles.
Anal. Chem. 2015, 87, 51675172. [CrossRef] [PubMed]
23. Luo, X.; Davis, J.J. Electrical biosensors and the label free detection of protein disease biomarkers.
Chem. Soc. Rev. 2013, 42, 59445962. [CrossRef] [PubMed]
24. Wu, Y.; Zhan, S.; Xu, L.; Shi, W.; Xi, T.; Zhan, X.; Zhou, P. A simple and label-free sensor for mercury
(ii) detection in aqueous solution by malachite green based on a resonance scattering spectral assay.
Chem. Commun. 2011, 47, 60276029. [CrossRef] [PubMed]
25. Blbl, G.; Hayat, A.; Andreescu, S. Ssdna functionalized nanoceria: A redox active aptaswitch for
biomolecular recognition. Adv. Healthc. Mater. 2016. [CrossRef] [PubMed]
26. Ellington, A.D. In vitro selection of rna molecules that bind specific ligands. Nature 1990, 346, 818822.
[CrossRef] [PubMed]
27. Rhouati, A.; Yang, C.; Hayat, A.; Marty, J.-L. Aptamers: A promising tool for ochratoxin a detection in food
analysis. Toxins 2013, 5, 19882008. [CrossRef] [PubMed]
28. Du, Y.; Li, B.; Wang, E. Fitting makes sensing simple: Label-Free detection strategies based on nucleic
acid aptamers. Acc. Chem. Res. 2012, 46, 203213. [CrossRef] [PubMed]
29. Gopinath, S.C.B.; Lakshmipriya, T.; Awazu, K. Colorimetric detection of controlled assembly and disassembly
of aptamers on unmodified gold nanoparticles. Biosens. Bioelectron. 2014, 51, 115123. [CrossRef] [PubMed]
30. De-los-Santos-lvarez, N.; Lobo-Castan, M.A.J.; Miranda-Ordieres, A.J.; Tun-Blanco, P. Aptamers as
recognition elements for label-free analytical devices. TrAC Trends Anal. Chem. 2008, 27, 437446. [CrossRef]
31. Citartan, M.; Gopinath, S.C.; Tominaga, J.; Tan, S.-C.; Tang, T.-H. Assays for aptamer-based platforms.
Biosens. Bioelectron. 2012, 34, 111. [CrossRef] [PubMed]
32. Neumann, O.; Zhang, D.; Tam, F.; Lal, S.; Wittung-Stafshede, P.; Halas, N.J. Direct optical detection of
aptamer conformational changes induced by target molecules. Anal. Chem. 2009, 81, 1000210006. [CrossRef]
[PubMed]
33. Hayat, A.; Yang, C.; Rhouati, A.; Marty, J.L. Recent advances and achievements in nanomaterial-based,
and structure switchable aptasensing platforms for ochratoxin a detection. Sensors 2013, 13, 1518715208.
[CrossRef] [PubMed]
34. Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Nucleic acid-functionalized pt nanoparticles: Catalytic labels
for the amplified electrochemical detection of biomolecules. Anal. Chem. 2006, 78, 22682271. [CrossRef]
[PubMed]
35. Nutiu, R.; Li, Y. Structure-Switching signaling aptamers: Transducing molecular recognition into fluorescence
signaling. Chem.A Eur. J. 2004, 10, 18681876. [CrossRef] [PubMed]
36. Rodriguez, M.C.; Kawde, A.-N.; Wang, J. Aptamer biosensor for label-free impedance spectroscopy detection
of proteins based on recognition-induced switching of the surface charge. Chem. Commun. 2005, 34,
42674269. [CrossRef] [PubMed]
37. Liu, S.-J.; Nie, H.-G.; Jiang, J.-H.; Shen, G.-L.; Yu, R.-Q. Electrochemical sensor for mercury (ii) based on
conformational switch mediated by interstrand cooperative coordination. Anal. Chem. 2009, 81, 57245730.
[CrossRef] [PubMed]
38. Cheng, A.K.; Sen, D.; Yu, H.-Z. Design and testing of aptamer-based electrochemical biosensors for proteins
and small molecules. Bioelectrochemistry 2009, 77, 112. [CrossRef] [PubMed]
39. Ho, H.-A.; Leclerc, M. Optical sensors based on hybrid aptamer/conjugated polymer complexes. J. Am.
Chem. Soc. 2004, 126, 13841387. [CrossRef] [PubMed]
Sensors 2016, 16, 2178 19 of 21

40. Feng, C.; Dai, S.; Wang, L. Optical aptasensors for quantitative detection of small biomolecules: A review.
Biosens. Bioelectron. 2014, 59, 6474. [CrossRef] [PubMed]
41. Feng, K.; Sun, C.; Kang, Y.; Chen, J.; Jiang, J.-H.; Shen, G.-L.; Yu, R.-Q. Label-Free electrochemical detection
of nanomolar adenosine based on target-induced aptamer displacement. Electrochem. Commun. 2008, 10,
531535. [CrossRef]
42. Xiao, Y.; Piorek, B.D.; Plaxco, K.W.; Heeger, A.J. A reagentless signal-on architecture for electronic,
aptamer-based sensors via target-induced strand displacement. J. Am. Chem. Soc. 2005, 127, 1799017991.
[CrossRef] [PubMed]
43. He, J.-L.; Wu, Z.-S.; Zhou, H.; Wang, H.-Q.; Jiang, J.-H.; Shen, G.-L.; Yu, R.-Q. Fluorescence aptameric sensor
for strand displacement amplification detection of cocaine. Anal. Chem. 2010, 82, 13581364. [CrossRef]
[PubMed]
44. Peng, Y.; Zhang, D.; Li, Y.; Qi, H.; Gao, Q.; Zhang, C. Label-Free and sensitive faradic impedance aptasensor
for the determination of lysozyme based on target-induced aptamer displacement. Biosens. Bioelectron. 2009,
25, 9499. [CrossRef] [PubMed]
45. Willner, I.; Zayats, M. Electronic aptamer-based sensors. Angew. Chem. Int. Ed. Engl. 2007, 46, 64086418.
[CrossRef] [PubMed]
46. Li, L.-D.; Zhao, H.-T.; Chen, Z.-B.; Mu, X.-J.; Guo, L. Aptamer biosensor for label-free impedance spectroscopy
detection of thrombin based on gold nanoparticles. Sens. Actuators B Chem. 2011, 157, 189194. [CrossRef]
47. Zhang, X.; Yadavalli, V.K. Surface immobilization of DNA aptamers for biosensing and protein interaction
analysis. Biosens. Bioelectron. 2011, 26, 31423147. [CrossRef] [PubMed]
48. Balamurugan, S.; Obubuafo, A.; Soper, S.A.; Spivak, D.A. Surface immobilization methods for aptamer
diagnostic applications. Anal. Bioanal. Chem. 2008, 390, 10091021. [CrossRef] [PubMed]
49. Samanta, D.; Sarkar, A. Immobilization of bio-macromolecules on self-assembled monolayers: Methods and
sensor applications. Chem. Soc. Rev. 2011, 40, 25672592. [CrossRef] [PubMed]
50. Fang, Y. Label-Free cell-based assays with optical biosensors in drug discovery. Assay Drug Dev. Technol.
2006, 4, 583595. [CrossRef] [PubMed]
51. Huang, Y.; Chen, J.; Zhao, S.; Shi, M.; Chen, Z.-F.; Liang, H. Label-Free colorimetric aptasensor based on
nicking enzyme assisted signal amplification and dnazyme amplification for highly sensitive detection of
protein. Anal. Chem. 2013, 85, 44234430. [CrossRef] [PubMed]
52. Li, T.; Dong, S.; Wang, E. Label-Free colorimetric detection of aqueous mercury ion (Hg2+ ) using
Hg2+ -modulated g-quadruplex-based dnazymes. Anal. Chem. 2009, 81, 21442149. [CrossRef] [PubMed]
53. Yang, C.; Lates, V.; Prieto-Simon, B.; Marty, J.L.; Yang, X. Aptamer-Dnazyme hairpins for biosensing of
ochratoxin a. Biosens. Bioelectron. 2012, 32, 208212. [CrossRef] [PubMed]
54. Tombelli, S.; Minunni, M.; Mascini, M. Analytical applications of aptamers. Biosens. Bioelectron. 2005, 20,
24242434. [CrossRef] [PubMed]
55. Vestergaard, M.; Kerman, K.; Tamiya, E. An overview of label-free electrochemical protein sensors. Sensors
2007, 7, 34423458. [CrossRef]
56. Chang, B.-Y.; Park, S.-M. Electrochemical impedance spectroscopy. Ann. Rev. Anal. Chem. 2010, 3, 207229.
[CrossRef] [PubMed]
57. Tamayo, J.; Kosaka, P.M.; Ruz, J.J.; San Paulo, .; Calleja, M. Biosensors based on nanomechanical systems.
Chem. Soc. Rev. 2013, 42, 12871311. [CrossRef] [PubMed]
58. Raiteri, R.; Grattarola, M.; Butt, H.-J.; Skldal, P. Micromechanical cantilever-based biosensors. Sens. Actuators
B Chem. 2001, 79, 115126. [CrossRef]
59. Janshoff, A.; Galla, H.J.; Steinem, C. Piezoelectric mass-sensing devices as biosensorsAn alternative to
optical biosensors? Angew. Chem. Int. Ed. Engl. 2000, 39, 40044032. [CrossRef]
60. Zain, M.E. Impact of mycotoxins on humans and animals. J. Saudi Chem. Soc. 2011, 15, 129144. [CrossRef]
61. Rai, M.; Jogee, P.S.; Ingle, A.P. Emerging nanotechnology for detection of mycotoxins in food and feed. Int. J.
Food Sci. Nutr. 2015, 66, 363370. [CrossRef] [PubMed]
62. Yang, C.; Lates, V.; Prieto-Simn, B.; Marty, J.-L.; Yang, X. Rapid high-throughput analysis of ochratoxin a by
the self-assembly of dnazymeaptamer conjugates in wine. Talanta 2013, 116, 520526. [CrossRef] [PubMed]
63. Wang, C.; Dong, X.; Liu, Q.; Wang, K. Label-Free colorimetric aptasensor for sensitive detection of ochratoxin
a utilizing hybridization chain reaction. Anal. Chim. Acta 2015, 860, 8388. [CrossRef] [PubMed]
Sensors 2016, 16, 2178 20 of 21

64. Zhang, J.; Zhang, X.; Yang, G.; Chen, J.; Wang, S. A signal-on fluorescent aptasensor based on Tb3+ and
structure-switching aptamer for label-free detection of ochratoxin a in wheat. Biosens. Bioelectron. 2013, 41,
704709. [CrossRef] [PubMed]
65. Lv, Z.; Chen, A.; Liu, J.; Guan, Z.; Zhou, Y.; Xu, S.; Yang, S.; Li, C. A simple and sensitive approach for
ochratoxin a detection using a label-free fluorescent aptasensor. PLoS ONE 2014, 9, e85968. [CrossRef]
[PubMed]
66. McKeague, M.; Velu, R.; Hill, K.; Bardczy, V.; Mszros, T.; DeRosa, M. Selection and characterization of a
novel DNA aptamer for label-free fluorescence biosensing of ochratoxin A. Toxins 2014, 6, 2435. [CrossRef]
[PubMed]
67. Lu, L.; Wang, M.; Liu, L.-J.; Leung, C.-H.; Ma, D.-L. Label-Free luminescent switch-on probe for ochratoxin a
detection using a g-quadruplex-selective iridium (iii) complex. ACS Appl. Mater. Interfaces 2015, 7, 83138318.
[CrossRef] [PubMed]
68. Park, J.-H.; Byun, J.-Y.; Mun, H.; Shim, W.-B.; Shin, Y.-B.; Li, T.; Kim, M.-G. A regeneratable, label-free,
localized surface plasmon resonance (lspr) aptasensor for the detection of ochratoxin A. Biosens. Bioelectron.
2014, 59, 321327. [CrossRef] [PubMed]
69. Prabhakar, N.; Matharu, Z.; Malhotra, B.D. Polyaniline langmuir-blodgett film based aptasensor for
ochratoxin a detection. Biosens. Bioelectron. 2011, 26, 40064011. [CrossRef] [PubMed]
70. Castillo, G.; Lamberti, I.; Mosiello, L.; Hianik, T. Impedimetric DNA aptasensor for sensitive detection of
ochratoxin a in food. Electroanalysis 2012, 24, 512520. [CrossRef]
71. Hayat, A.; Sassolas, A.; Marty, J.-L.; Radi, A.-E. Highly sensitive ochratoxin a impedimetric aptasensor based
on the immobilization of azido-aptamer onto electrografted binary film via click chemistry. Talanta 2013, 103,
1419. [CrossRef] [PubMed]
72. Mishra, R.K.; Hayat, A.; Catanante, G.; Ocaa, C.; Marty, J.-L. A label free aptasensor for ochratoxin A
detection in cocoa beans: An application to chocolate industries. Anal. Chim. Acta 2015, 889, 106112.
[CrossRef] [PubMed]
73. Hayat, A.; Andreescu, S.; Marty, J.-L. Design of peg-aptamer two piece macromolecules as convenient
and integrated sensing platform: Application to the label free detection of small size molecules.
Biosens. Bioelectron. 2013, 45, 168173. [CrossRef] [PubMed]
74. Hayat, A.; Haider, W.; Rolland, M.; Marty, J.-L. Electrochemical grafting of long spacer arms of
hexamethyldiamine on a screen printed carbon electrode surface: Application in target induced ochratoxin a
electrochemical aptasensor. Analyst 2013, 138, 29512957. [CrossRef] [PubMed]
75. Evtugyn, G.; Porfireva, A.; Stepanova, V.; Kutyreva, M.; Gataulina, A.; Ulakhovich, N.; Evtugyn, V.;
Hianik, T. Impedimetric aptasensor for ochratoxin a determination based on au nanoparticles stabilized with
hyper-branched polymer. Sensors 2013, 13, 1612916145. [CrossRef] [PubMed]
76. Karimi, A.; Hayat, A.; Andreescu, S. Biomolecular detection at ssdna-conjugated nanoparticles by
nano-impact electrochemistry. Biosens. Bioelectron. 2017, 87, 501507. [CrossRef] [PubMed]
77. Luan, Y.; Chen, J.; Xie, G.; Li, C.; Ping, H.; Ma, Z.; Lu, A. Visual and microplate detection of aflatoxin b2 based
on nacl-induced aggregation of aptamer-modified gold nanoparticles. Microchim. Acta 2015, 182, 9951001.
[CrossRef]
78. Luan, Y.; Chen, Z.; Xie, G.; Chen, J.; Lu, A.; Li, C.; Fu, H.; Ma, Z.; Wang, J. Rapid visual detection of aflatoxin
b1 by label-free aptasensor using unmodified gold nanoparticles. J. Nanosci. Nanotechnol. 2015, 15, 13571361.
[CrossRef] [PubMed]
79. Seok, Y.; Byun, J.-Y.; Shim, W.-B.; Kim, M.-G. A structure-switchable aptasensor for aflatoxin b1 detection
based on assembly of an aptamer/split dnazyme. Anal. Chim. Acta 2015, 886, 182187. [CrossRef] [PubMed]
80. Shim, W.-B.; Mun, H.; Joung, H.-A.; Ofori, J.A.; Chung, D.-H.; Kim, M.-G. Chemiluminescence competitive
aptamer assay for the detection of aflatoxin b1 in corn samples. Food Control 2014, 36, 3035. [CrossRef]
81. Chen, J.; Zhang, X.; Cai, S.; Wu, D.; Chen, M.; Wang, S.; Zhang, J. A fluorescent aptasensor based on
DNA-scaffolded silver-nanocluster for ochratoxin a detection. Biosens. Bioelectron. 2014, 57, 226231.
[CrossRef] [PubMed]
82. Nguyen, B.H.; Dai Tran, L.; Do, Q.P.; Le Nguyen, H.; Tran, N.H.; Nguyen, P.X. Label-Free detection of
aflatoxin m1 with electrochemical Fe3 O4 /polyaniline-based aptasensor. Mater. Sci. Eng. C 2013, 33,
22292234. [CrossRef] [PubMed]
Sensors 2016, 16, 2178 21 of 21

83. Istambouli, G.; Paniel, N.; Zara, L.; Granados, L.R.; Barthelmebs, L.; Noguer, T. Development of an
impedimetric aptasensor for the determination of aflatoxin m1 in milk. Talanta 2016, 146, 464469. [CrossRef]
[PubMed]
84. Goud, K.Y.; Catanante, G.; Hayat, A.; Satyanarayana, M.; Gobi, K.V.; Marty, J.L. Disposable and portable
electrochemical aptasensor for label free detection of aflatoxin b1 in alcoholic beverages. Sens. Actuators
B Chem. 2016, 235, 466473. [CrossRef]
85. Castillo, G.; Spinella, K.; Poturnayov, A.; nejdrkov, M.; Mosiello, L.; Hianik, T. Detection of aflatoxin b1
by aptamer-based biosensor using pamam dendrimers as immobilization platform. Food Control 2015, 52,
918. [CrossRef]
86. Chen, X.; Huang, Y.; Ma, X.; Jia, F.; Guo, X.; Wang, Z. Impedimetric aptamer-based determination of the
mold toxin fumonisin b1. Microchim. Acta 2015, 182, 17091714. [CrossRef]
87. Chen, X.; Bai, X.; Li, H.; Zhang, B. Aptamer-Based microcantilever array biosensor for detection of fumonisin
b-1. RSC Adv. 2015, 5, 3544835452. [CrossRef]
88. Amaya-Gonzlez, S.; de-los-Santos-lvarez, N.; Miranda-Ordieres, A.; Lobo-Castan, M. Aptamer-Based
analysis: A promising alternative for food safety control. Sensors 2013, 13, 1629216311. [CrossRef] [PubMed]
89. Cruz-Aguado, J.A.; Penner, G. Determination of ochratoxin a with a DNA aptamer. J. Agric. Food Chem. 2008,
56, 1045610461. [CrossRef] [PubMed]
90. Le, L.C.; Cruz-Aguado, J.A.; Penner, G.A. Dna ligands for aflatoxin and zearalenone. U.S. Patent 13/391,426,
6 September 2012.
91. Ma, X.; Wang, W.; Chen, X.; Xia, Y.; Duan, N.; Wu, S.; Wang, Z. Selection, characterization and application of
aptamers targeted to aflatoxin b2. Food Control 2015, 47, 545551. [CrossRef]
92. Malhotra, S.; Pandey, A.; Rajput, Y.; Sharma, R. Selection of aptamers for aflatoxin m1 and their
characterization. Jo. Mol. Recognit. 2014, 27, 493500. [CrossRef] [PubMed]
93. McKeague, M.; Bradley, C.R.; Girolamo, A.D.; Visconti, A.; Miller, J.D.; DeRosa, M.C. Screening and initial
binding assessment of fumonisin b1 aptamers. Int. J. Mol. Sci. 2010, 11, 48644881. [CrossRef] [PubMed]
94. Chen, X.; Huang, Y.; Duan, N.; Wu, S.; Xia, Y.; Ma, X.; Zhu, C.; Jiang, Y.; Ding, Z.; Wang, Z. Selection and
characterization of single stranded DNA aptamers recognizing fumonisin b1. Microchim. Acta 2014, 181,
13171324. [CrossRef]
95. Chen, X.; Huang, Y.; Duan, N.; Wu, S.; Ma, X.; Xia, Y.; Zhu, C.; Jiang, Y.; Wang, Z. Selection and identification
of ssdna aptamers recognizing zearalenone. Anal. Bioanal. Chem. 2013, 405, 65736581. [CrossRef] [PubMed]
96. Ruscito, A.; Smith, M.; Goudreau, D.N.; DeRosa, M.C. Current status and future prospects for aptamer-based
mycotoxin detection. J. AOAC Int. 2016, 99, 865877. [CrossRef] [PubMed]
97. Zhou, J.; Rossi, J. Aptamers as targeted therapeutics: Current potential and challenges. Nat. Rev. Drug Discov.
2016. [CrossRef] [PubMed]
98. Mascini, M. Aptamers in Bioanalysis; Wiley: Florence, Italy, 2009.
99. Eaton, B.E.; Gold, L.; Hicke, B.J.; Janji, N.; Jucker, F.M.; Sebesta, D.P.; Tarasow, T.M.; Willis, M.C.; Zichi, D.A.
Post-selex combinatorial optimization of aptamers. Bioorgan. Med. Chem. 1997, 5, 10871096. [CrossRef]

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