Iron and iron-oxide magnetic nanoparticles as signal

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Trends in Analytical Chemistry 72 (2015) 1–9
Contents lists available at ScienceDirect
Trends in Analytical Chemistry
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a c
Iron and iron-oxide magnetic nanoparticles as signal-amplification
elements in electrochemical biosensing
Mohammad Hasanzadeh a,*, Nasrin Shadjou b,c,**, Miguel de la Guardia d
a
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51664, Iran
Department of Nanochemistry, Nano Technology Center, Urmia University, Urmia, Iran
Department of Chemistry, Faculty of Chemistry, Urmia University, Urmia, Iran
d Department of Analytical Chemistry, University of Valencia, Dr. Moliner 50, Burjassot, 46100 Valencia, Spain
b
c
A R T I C L E
I N F O
Keywords:
Antigen-antibody interaction
Biological molecule
Biomarker
Electrochemical biosensing
Enzyme
Immunosensor
Iron oxide
Magnetic bead
Magnetic nanoparticle
Signal amplification
A B S T R A C T
Growing demands for ultrasensitive biosensing have led to the development of numerous signalamplification strategies. Based on their unique properties (i.e., electro-conductivity, bio-compatibility and
ease of synthesis), various iron magnetic nanoparticles (MNPs) have proved to be an excellent nanomaterial
for applications in electrochemical biosensing. This review shows how iron MNPs have made significant contributions in the development of electrochemical nanobiosensors, including immuno-, enzyme,
DNA and aptamer types. More importantly, we discuss in detail different aspects of the electrochemical
biosensors (e.g., modes of magnetic particles, detection techniques, analytes and the corresponding sensitivity and sample matrix, and several prominent characteristics). Accordingly, we discuss research
opportunities and future development trends in these areas.
© 2015 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
4.
Introduction .............................................................................................................................................................................................................................................................
1.1.
Principles of biosensor function ..........................................................................................................................................................................................................
1.2.
Electrochemical biosensing ...................................................................................................................................................................................................................
Magnetic nanoparticles in electrochemical biosensing ............................................................................................................................................................................
2.1.
Why nanoparticles? .................................................................................................................................................................................................................................
2.2.
Why magnetic nanoparticles? .............................................................................................................................................................................................................
2.3.
Why nanomaterial-based electrochemical-signal amplification? ...........................................................................................................................................
2.4.
Why iron-based magnetic nanoparticles in electrochemical biosensing? ...........................................................................................................................
Types of magnetic nanoparticle in electrochemical biosensing ............................................................................................................................................................
3.1.
Iron and iron-oxide magnetic nanoparticle-based electrochemical enzymatic biosensors ...........................................................................................
3.2.
Iron and iron-oxide magnetic nanoparticle-based electrochemical DNA biosensing ......................................................................................................
3.3.
Iron and iron-oxide magnetic nanoparticle-based electrochemical immunosensors ......................................................................................................
Conclusion and outlook .......................................................................................................................................................................................................................................
Acknowledgments .................................................................................................................................................................................................................................................
References ................................................................................................................................................................................................................................................................
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1. Introduction
1.1. Principles of biosensor function
* Corresponding author. Tel.: +98 914 3619877; Fax: +98 41133632312.
E-mail address: [email protected], [email protected]
(M. Hasanzadeh).
** Corresponding author.
E-mail address: [email protected], [email protected] (N. Shadjou).
http://dx.doi.org/10.1016/j.trac.2015.03.016
0165-9936/© 2015 Elsevier B.V. All rights reserved.
Biosensing is based on immobilized biomolecules for detection and determination of target analytes. Such sensing biomolecules
should be attached to the surface of a signal transducer. After interaction, the biological-recognition event generates an optical or
electrical signal. Thus, biomolecule immobilization plays a crucial
role in achieving biosensing with extended device lifetime. The substrate materials [e.g., electrodes, mesoporous materials, nanoparticles
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M. Hasanzadeh et al./Trends in Analytical Chemistry 72 (2015) 1–9
(NPs), nanotubes, and graphene (GN)] for biomolecule immobilization must be modified to introduce functional groups that are
attached to biomolecules with high bonding strength, excellent longterm stability, biocompatibility, and high activity [1]. In general, a
biosensor is an analytical device composed of two components, a
bioreceptor and a transducer. First, the bioreceptor is a biomolecule
that recognizes the target analyte, and, second, the transducer converts the recognition event into a measurable signal [2].
Significantly, a great deal of research on preparing functional materials for electrode construction are extending the applications of
electrochemical biosensors. For example, Walcarius et al. highlighted
recent advances of nanostructured materials in the rational design of
bioelectrodes and related biosensing systems [13]. The appeal of
nanomaterials lies in their capability to act as an immobilization matrix
and their exclusive features. These properties, combined with the
functioning of biomolecules, contribute to the improvement of
bioelectrode performance in terms of sensitivity and specificity.
1.2. Electrochemical biosensing
Electrochemical biosensors combine the analytical power of electrochemical techniques with the specificity of biological-recognition
processes [3–7]. The basic principle of electrochemical biosensors
is that chemical reaction between immobilized biomolecule and
target analyte produces or consumes ions or electrons, which affects
the measurable electrical properties of the solution (e.g., electric
current or potential). In biochemistry or electrochemistry, the reaction would generate a measurable current (amperometric), a
measurable potential or charge accumulation (potentiometric) or
alter the impedance (both resistance and reactance) of the medium
between the electrodes [8].
2. Magnetic nanoparticles in electrochemical biosensing
2.1. Why nanoparticles?
The appearance of nanoscience is opening novel fields for the
application of NPs in biosensing technology. NPs are of great interest in the world of nanoscience based on their physical and
chemical properties. Such properties offer excellent prospects for
chemical and biological sensing [9]. NPs were widely used in recent
years as useful, sensitive tools for the electronic, optical, and
microgravimetric transduction of different biomolecular recognition events [10]. NPs can therefore be seriously enhanced by
incorporating them within biological systems. The signal enhancement associated with the use of NP-amplifying labels and with the
formation of NP-biomolecule assemblies provides the basis for sensitive electrical and optical detection. Such procedures couple the
extensive features of NP-biomolecule associations with sensitive electrochemical or optical transduction. In general, NP-based biosensors
offer excellent potential for diagnostics and can have a deep impact
upon bio-analytical applications.
2.2. Why magnetic nanoparticles?
The capability of magnetic NPs (MNPs) to provide biocompatibility is a benefit for the preparation of bio-(sensors). However, MNPs
allow electron transfer between redox systems and bulk-electrode
materials based on their unique properties (e.g., high surface energy
and large surface area and functioning as electron-conducting
pathways) [11]. MNPs have also established useful interfaces for the
electrocatalysis of redox processes of molecules (e.g., H 2 O 2 ,
O2 or NADH) involved in many significant biochemical reactions
[12].
2.3. Why nanomaterial-based electrochemical-signal amplification?
With notable attainments in nanoscience, nano-sized materialbased electrochemical-signal amplifications have excellent potential to
improve both sensitivity and selectivity of electrochemical biosensors.
It is recognized that the electrode materials play a serious role in preparing high-performance electrochemical-sensing platforms for
detecting target molecules. Also, nanomaterials produce a synergistic
effect for conductivity and biocompatibility to accelerate signal transduction but also to amplify recognition events with designed signal tags.
2.4. Why iron-based magnetic nanoparticles in electrochemical
biosensing?
The applications of NPs in biosensors can be classified into two
categories according to their functions:
•
•
NP-modified transducers for bioanalytical applications; and,
biomolecule-NP conjugates as labels for biosensing and bioassays.
We intend to survey some major advances and milestones in biosensor development based upon NP labels and their roles in
biosensors and bioassays for nucleic acids and proteins. Moreover,
we focus on some of the key fundamental properties of certain NPs
that make them ideal for different biosensing applications.
Electrochemical immunosensors, enzyme sensors, and tissue and
DNA biosensors are designed through immobilizing on the working
electrode surface biological-recognition elements of antibodies (Abs),
enzyme, tissue and DNA, respectively [14]. The NPs could be immobilized on the surface of the transducers (e.g., physical adsorption,
chemical-covalent bonding, or electrodeposition) for electrochemicalsignal generation and amplification [15]. Iron-based MNPs provide
a large surface area to immobilize as many biomolecules as
possible, resulting in a lower limit of detection (LOD). Moreover, ironbased MNPs can play roles in concentration and purification. Ironbased MNPs are particularly efficient in detecting analytes in
complex sample matrices, which may exhibit either poor mass transport to the biosensor or physical blockage of the biosensor surface
by non-specific adsorption [16]. Iron-based MNPs can remove the
need for sample pretreatment by centrifugation or chromatography, thus shortening the handling time [17]. Also, most iron-based
MNPs, especially iron oxides, are biocompatible and non-genotoxic;
they can be applied for simple adsorption of biomolecules,
functionalized or encapsulated in polymers, metal or silica NPs, or
carbon materials to enhance the biocompatibility and increase the
functionalities [18]. Thus, iron-based MNPs provide a promising experimental platform for developing both types of electrochemical
biosensor.
This review emphasizes more recent progress in electrochemical sensors, biosensors and immunosensors, based on iron and ironoxide MNPs (i.e., in the period January 2014 to January 2015). The
purpose of this review is to illustrate new progress in areas ranging
from novel electroanalytical techniques to electrochemical-signal
amplification based on iron and iron-oxide MNPs. We also attempt
to introduce the significant development of electrochemical
biosensors based on iron and iron-oxide MNPs. Therefore, based on
the previous review articles [14,19] and selected research articles
from January 2014 to January 2015, we comprehensively summarize various iron and iron-oxide MNP-based electrochemical
biosensors including immunosensors, enzyme sensors, DNA sensors
and aptamer sensors. More importantly, we discuss in detail different aspects of the biosensors (e.g., modes of MNPs, injection and
detection techniques, labels, analytes and the corresponding sample
matrix, and sensitivity). Consequently, we discuss several outstanding properties of the biosensors, research opportunities and the
development potential and prospects.
M. Hasanzadeh et al./Trends in Analytical Chemistry 72 (2015) 1–9
3. Types of magnetic nanoparticle in electrochemical
biosensing
3.1. Iron and iron-oxide magnetic nanoparticle-based
electrochemical enzymatic biosensors
In recent years, there was an increasing trend in the design and
the development of MNPs towards enzymatic biosensing [20,21].
MNPs are an interesting material for the immobilization of desired
enzymes because of some superior properties (i.e., electroconductivity, biocompatibility and ease of synthesis) [22].
Enzyme-immobilized MNPs could potentially lead to unique
properties (e.g., large surface area, high bioactivity and excellent stability) [23,24]. Iron and iron-oxide MNPs have been widely employed
in electrochemical biosensors as nanosized supports for the immobilization of analytical biomolecules. In particular, the
immobilization of enzymes on the surface of these NPs offers numerous advantages, including enhancement of the enzymatic activity
and reduction of the mass-transfer processes associated with recognition of substrates by enzymes. For example, a novel laccase
biosensor was fabricated by entrapping laccase in GN-chitosan composite materials and applying them to determine hydroquinone [25].
In this work, according to a synergistic effect between GN and
chitosan, GN-chitosan-Fe 2 O 3 composites exhibit robust filmforming ability and good electrical conductivity. An embedding
procedure immobilizes laccase into the composite film without a
cross-linking reagent. This biosensor catalyzed the oxidation of hydroquinone to p-quinone, and p-quinone back to hydroquinone.
In another report, a novel phenolic biosensor was prepared on the
basis of a composite of polydopamine (PDA)-laccase (Lac)-Ni/Fe NPloaded carbon nanofibers (NiCNFs) [26]. In this work, NiCNFs were
fabricated by a combination of electrospinning and high-temperature
carbonization. Subsequently, the magnetic composite was obtained
through one-pot Lac-catalyzed oxidation of dopamine (DA) in an
aqueous suspension containing Lac, NiCNFs, and DA. Finally, a magnetic glassy-carbon electrode (MGCE) was employed to separate and
to immobilize the composite; the modified electrode was then
denoted as PDA-Lac-NiCNFs/MGCE. As shown in Fig. 1, it can be clearly
seen that the short NiCNFs dispersed well in the PDA-Lac-NiCNFs composite. Most of them were embedded in the composite, and would
play the role of “molecular wires”, connecting the active center of
Lac and the surface of the MGCE. The composite formed a membrane on the surface of electrode. Besides, there were some “pores”
or “ravines” existing in the membrane, which were beneficial to the
diffusion of substrate. According to results obtained by Li and
Fig. 1. SEM images of the surface of PDA-Lac-NiCNFs/MGCE [26].
3
co-workers [26], the PDA-Lac-NiCNFs/MGCE for biosensing of catechol had an LOD of 0.69 μM, and a linear range of 0.001–9.1 mM.
Iron-oxide NPs also provide a favorable microenvironment for
electrochemical devices where enzymes may exchange electrons directly with the transducer, improving the sensitivity and the selectivity
of electrochemical biosensors. Recently, Martin and co-workers [27]
reported a novel biosensor based on core-shell [email protected](dopamine)
MNPs, which were prepared through an in situ self-polymerization
method. According to this report, the core-shell NPs were employed as solid supports for the covalent immobilization of
horseradish peroxidase (HRP), and the resulting biofunctionalized
MNPs were employed to construct an amperometric biosensor for
H2O2. This enzyme biosensor had a low LOD of 182 nM.
Compared to immobilizing enzymes onto a substrate surface, incorporating enzymes into matrix has the potential to increase the
enzyme loading and to protect the enzyme from the surrounding
environment. For this purpose, Qu et al. [28], prepared a novel biosensor based on immobilizing glucose oxidase on hydrogel. To increase
the loading of glucose oxidase (GOx) and simplify glucose-biosensor
fabrication, hydrogel prepared from ferrocene (Fc)-modified aminoacid phenylalanine (Phe, F) was utilized for the incorporating GOx.
According to this report, the synthesized hydrogel contained a significant number of Fc moieties, which could be considered as an
ideal matrix to immobilize enzymes for preparing mediator-based
biosensors. It seems that the favorable network structure and good
biocompatibility of the hydrogel could effectively avoid enzyme
leakage and maintain the bioactivity of the enzymes, which resulted in good stability of the biosensor. Interestingly, this research
group used this biosensor to detect glucose in blood samples with
results comparable to those obtained from the hospital.
Also, in cholesterol biosensors, a novel sensitive, selective biosensor based on cholesterol oxidase co-immobilized with α-Fe2O3 micropine-shaped hierarchical structures was prepared by Umar et al. [29].
The results showed that α-Fe2O3 micro-pine-shaped hierarchical structures can be used to fabricate a highly sensitive, selective cholesterol
biosensor. But the reported LOD meant this approach is unsuitable.
The development of electrochemical biosensors to detect various
pesticides was an active research area in the past decade. Most electrochemical pesticide biosensors are based on inhibiting enzyme
acetyl cholinesterase (AChE) [30,31]. The main approach used to
measure this inhibition is based on the amperometric/voltammetric
detection of thiocholine, which is the enzymatic reaction product
of acetylthiocholine by oxidation at a constant potential at the electrode. The equations below show this reaction.
Recently, Jeyapragasam and Saraswathi [32] developed an
AChE enzyme biosensor for carbofuran based on the Fe3O4-CH
nanocomposite formed by a simple solution-mixing process. These
researchers demonstrated that the distinct physico-chemical properties of Fe3O4 and CH can help the effective immobilization of the
AChE enzyme onto the nanocomposite. The presence of CH especially prevents not only the aggregation of the MNPs but also the
loss of the enzyme molecules by providing a biocompatible microenvironment to maintain enzyme activity. The SEM image of AChE/
Fe3O4-CH (Fig. 2) shows reduced porosity with large particles due
to coverage by the enzyme. The Fe3O4-CH nanocomposite shows good
electrocatalysis in oxidizing thiocholine, leading to development of
a highly sensitive, reliable biosensor for real sample analysis.
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M. Hasanzadeh et al./Trends in Analytical Chemistry 72 (2015) 1–9
Fig. 2. SEM images of the AChE/Fe3O4-CH [32].
In concluding this section, it is important to point out that, although great advances in enzymatic biosensors based on iron MNPs
have been achieved, there are many challenges in developing a
biosensing system for real sample analysis. So far, almost all research on iron-MNP-based enzymatic biosensors was in the
laboratory. As for practical applications, few enzymatic biosensors
appear to be commercially feasible, apart from some bloodglucose and hand-held immunosensors.
No commercial enzymatic biosensor based on MNPs has been
reported so far, so, to commercialize enzymatic biosensors based
on iron MNPs, efforts need to be made to break some key technical barriers (e.g., controlling the morphology of iron MNPs on device,
realizing efficient enzyme immobilization, keeping enzyme bioactive
in the long term, and reducing matrix interference and sensor
fouling).
First, controlling the morphologies of iron MNPs for biosensor
design is necessary because the morphology of the nanosized material is one of the most important factors to determine the properties
for biosensor applications. Iron MNPs usually demonstrate sizedependent, shape-dependent and structure-dependent optical,
electronic, thermal, and structural properties.
Many synthesis methods have been developed for controllable
preparation of iron MNPs, but, for practical biosensor design, the
procedure of controlling synthesis and then depositing the NPs on
device is not a good choice because of the tendency towards aggregation of NPs. Thus, in-situ synthesis (e.g., electrochemical
deposition, CVD snd template synthesis) might be powerful tools
to control growth of iron MNPs on device directly.
Second, how to realize efficient enzyme immobilization is a very
critical question that should be addressed in commercializing
biosensors. Conventional strategies, including physical or chemical
immobilization, fall short in controlling the orientation of the enzyme
in order to implement efficient enzyme immobilization and to retain
the long-term bioactivity of enzyme. Recently, the coupling of sitedirected mutagenesis and immobilization proved a very useful
strategy for controlling the orientation of the enzyme and improving enzyme features from activity to stability. It should be further
studied to test whether or not site-directed mutagenesis and immobilization enzymes in iron MNPs could improve enzyme features.
Third, for practical application of iron-MNP-based enzymatic
biosensors, problems associated with matrix interference and sensor
fouling should be resolved. Biosensors might perform well under
controlled environments or with laboratory samples, but, in real
sample analysis, matrix interference and sensor fouling are detrimental factors for commercialization. A large surface area for NPs
might lead to more serious sensor fouling, so more effort should
be made to solve these problems.
Despite these numerous challenges, the iron-MNP-based enzymatic biosensing system is one of the most promising tools (e.g.,
food-safety detection, environmental-pollution control, and pointof-care testing). With the advancement of nanotechnology and
biotechnology, more enzymatic biosensing platforms based on iron
MNPs will be established and new breakthroughs in practical applications will be realized.
However, NPs have emerged as versatile tools for generating excellent supports for enzyme stabilization due to their small size and
large surface area. By proper surface modification, various iron MNPs
have been synthesized and successfully utilized for protein/
enzyme immobilization, and have displayed promise in practical
applications. We summarized in this sub-section the applications
of iron MNPs in enzyme immobilization, protein separation/
purification, medical science and food analysis. The immobilized
proteins/enzymes generally show better stability towards pH and
heat than free proteins/enzymes and can be recovered and reused
many times. However, the activity of some enzymes decreases to
some extent after immobilization, which indicates that more efforts
are required to explore immobilization techniques.
3.2. Iron and iron-oxide magnetic nanoparticle-based
electrochemical DNA biosensing
Deoxyribonucleic acid (DNA) is the carrier of genetic information and the foundation material of biological heredity. Sensing
specific DNA sequences plays a very important role in clinical diagnostics, gene therapy, food safety, environmental analysis, and
biodefense applications [33]. Since Watson and Crick proposed the
double-helix structure of DNA in 1953 [34,35], many researchers
have devoted attention to DNA sensors. Over the past decades, to
meet the growing demands for ultrasensitive detection, researchers developed many techniques to enhance the response of DNAbased electrochemical sensing by modifying DNA with different
functional materials.
With the continual development of nanotechnology, great attention has been paid to different metal NPs [36,37]. Because of
electroconductivity, biocompatibility and ease of synthesis, MNPs
have really improved the analytical performance to obtain the amplified detection signal and the stabilized recognition probes or
biosensing interface [38]. In this sub-section, we summarize the latest
developments in applying MNPs as signal-amplification elements
in ultrasensitive DNA-based electrochemical detection. We review
briefly various methods and different signal-amplification routes.
MNPs modified with different recognition elements have been
used for specific bioaffinity capture of different molecules. Alocilja
et al. [39] reported a highly-amplified electrochemical biosensor for
the simultaneous detection of multiple pathogens by using
nanotracers (PbS and CdS) terminating biobarcoded gold NPs (AuNPs)
and MNPs for easy, clean separation. After hybridization with the
target DNA, a magnetic field was used to separate the sandwich
complex from the unreacted reagents, leading to magnetic extraction and concentration. Then, the NP tracers were dissolved in 1 M
nitric acid, and the Pb2+ and Cd2+ ions released were detected by
square wave anodic stripping voltammetry on screen-printed carbonelectrode chips. This multiplex sensor obtained very low LODs.
Recently, some disposable magnetic DNA-based sensors with high
sensitivity were reported. In Pingarrón’s work, combining magnetic beads for DNA isolation and hybridization and an HRP-amplified
detection strategy, their magnetic hybridization DNA sensors showed
high sensitivity even without polymerase chain reaction (PCR) amplification [40]. However, this kind of biosensor has some obvious
limitations (e.g., the strength and the direction of the magnetic field
cannot be regulated due to the utilization of permanent magnet;
M. Hasanzadeh et al./Trends in Analytical Chemistry 72 (2015) 1–9
the hybridization and detection processes are separately performed, making the biosensor unsuitable for on-line analysis; and,
the high cost of analysis due to use of a single-use screen-printed
electrode and low discrimination ability for the single-base mismatch cannot be avoided).
To solve these limitations, a novel magnetically-controllable electrochemical biosensor based on a home-made electrically magneticcontrollable gold electrode (EMC-GE) has been reported for detection
of single-nucleotide polymorphisms (SNPs) [41]. The EMC-GE
combined the merits of heated electrode and magnetic electrode,
had notable advantages (e.g., strength and direction of the magnetic field and the temperature of the electrode surface could be
regulated according to the experimental strategy). These features
meant that the biosensor had ultra-high sensitivity by combining
MNP-based enzymatic catalysis amplification, better discrimination for single-base mismatch by controlling the temperature of the
electrode surface, and short analysis time, since the separation and
detection could be simultaneously performed, and low analysis cost,
due to the good reusability of the EMC-GE. It could be used to detect
down to 0.37 fM of target DNA with a satisfactory recovery and reproducibility, to determine the genotype single-base mismatch with
a discrimination factor of 1.47–2.63.
Interestingly, a DNA/chitosan-Fe3O4-MNP bio-complex film was
constructed for the immobilization of horseradish peroxidase (HRP)
on a GCE [42]. In this work, HRP was simply mixed with DNA,
chitosan and Fe3O4-NPs, and then applied to the electrode surface
to form an enzyme-incorporated polyion complex film. In this work,
direct electron transfer and electrocatalysis of HRP based on the DNA/
chitosan/Fe3O4 film were studied. The sensor prepared was used for
electrocatalytic reduction behavior towards H2O2. The reported LOD
for H2O2 measurement was 1 μM.
Also, a novel, sensitive electrochemical biosensor for selective
determination of DNA has been developed based on [email protected] involving 2-aminoethanethiol (AET)-functionalized graphene oxide
(GO) ([email protected]). In this report, first, 5-TA CCG GGT GCT
CGA GCT-(CH2)3-SH-3 single-stranded probe (ss-DNA) has been
was immobilized on [email protected] nanocomposite to form
[email protected] [43]. As shown in Fig. 3, morphology characterization demonstrated that the [email protected] are spherical with an
average diameter of 10–25 nm on a lighter shaded substrate corresponding to the planar GO sheet. It is uniformly distribution on
the GO sheet. Square-wave voltammetry (SWV) has been applied
to monitor the DNA hybridization by Basic Blue 41 (BB41) as an electrochemical indicator. As reported, the biosensor showed high
selectivity to one-base, two-base and three-base mismatched DNA.
The reported LOD for H2O2 measurement was 20 pM.
Fig. 3. TEM image of the [email protected] nanomaterial [43].
5
More recently, Chen et al. [44] prepared a lipase-based electrochemical biosensor for the quantitative determination of target DNA.
It was based on a stem-loop nucleic-acid probe labeled with ferrocene containing a butanoate ester that was hydrolyzed by lipase.
The other end of the probe DNA was linked, via carboxy groups, to
MNPs. The binding of target DNA transformed the hairpin structure of the probe DNA and caused exposure of ester bonds. The
quantity of target DNA in the concentration range was 0.001–10 nM,
determined by measuring the electrochemical current. This report
found that lipase could be applied for the detection of target DNA
in the future. Also, it does not require complicated conjugation chemistry and tedious label modification, so it may provide a general
platform for the detection of other target DNA.
Growing demands for ultrasensitive bioassays have led to the development of numerous signal-amplification strategies. Based on
their unique properties, iron and iron-oxide MNPs have been widely
used as signal-amplification elements in electrochemical DNAbased sensing. They can act as electrode materials to give significant
signal amplification. In addition, multiple amplification strategies,
which utilize two or more types of nanomaterial and amplification method, can further enhance analytical performance and offset
the insufficiency of each individual component.
However, there are still many challenges in the applications of
nanomaterials in the DNA-based electrochemical sensing:
(1) means to prepare monodispersed nanomaterials functionalized
with DNA, enzymes or other electroactive species;
(2) ways to exclude the non-specific adsorption of coexisting
species on the nanomaterials in the complex matrix, due to
the large surface area of nanomaterials; and,
(3) controllable synthesis of nanomaterials with repeatable structure and physicochemical properties in different batches.
Although iron MNPs have been widely used in DNA-based electrochemical sensing, tremendous opportunities still exist. Novel
iron MNPs conjugated with polymers fabricated recently should
be very useful for DNA-based electrochemical-sensing systems.
For example, porous ZIF-8 nanopolyhedra and related carbon
nanopolyhedra, which have a large surface area and pore volume,
may be explored as new electrode materials or carriers to amplify
the response signals in DNA-based electrochemical sensors. Also,
GN-related nanomaterials, which show superior electrocatalytic
performance, may act as catalysts to construct DNA-based electrochemical sensing system. Moreover, electrochemiluminescent and
photoelectrochemical assays, which have the advantages of both
electrochemical and optical detection, should be a promising direction in ultrasensitive DNA-based sensors with iron MNPs.
It is also interesting to integrate individual iron MNP-based DNA
electrochemical sensors into arrays for automatic bioanalysis and
high-throughput multi-analyte detection and to develop suitable
MNP-based DNA sensors for in vivo studies. With the persistent development of nanotechnology, we envisage that nanomaterials with
excellent biological and electrochemical properties will play very
important roles in constructing the ultrasensitive DNA-based electrochemical bioassays or nanodevices for biodetection at the singlecell and molecule level and for point-of-care (POC) diagnosis.
Taken together, we conclude that iron-MNP-based electrochemical DNA biosensors are suitable for detecting DNA hybridization
using electrochemical signals. The detection of specific DNA sequences by iron MNPs would benefit disease diagnosis. The
DNA nanobiosensor constructed using different immobilization
methods exhibited fast response, good selectivity, good sensitivity,
broad linear range and the best LODs. Although the LOD for DNAhybridization detection was suitable for applying to iron-MNPbased electrochemical biosensors, fabrication of MNPs can be easy
to reproduce.
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Although the fast advancement of iron-MNP-based electrochemical DNA biosensors highlights their future applications in
diverse scientific fields, the major impact appears to be in early detection of diseases, pathogens, genetic mutations and biotargets [45].
Also, iron-MNP-based electrochemical DNA biosensors provide fast,
simple, sensitive detection systems for pathogens and toxins and
may provide robust tools for forensic medicine and biodefense research. Nevertheless, some electrochemical-sensing platforms (e.g.,
CombiMatrix Electra-Sense) have already been commercialized.
3.3. Iron and iron-oxide magnetic nanoparticle-based
electrochemical immunosensors
Electrochemical-affinity sensors based on Abs offer great selectivity and sensitivity for early cancer diagnosis. Electrochemical
immunosensors offer a number of significant advantages, including high sensitivity, low cost, and ease of miniaturization, compared
with several other types of immunosensors based on fluorescence, chemiluminescence, surface-plasmon resonance, or quartzcrystal microbalance.
Recently, functionalized NPs, especially functionalized ironoxide NPs (Fe3O4-NPs) attracted much attention in the fabrication
of bio-sensing systems, due to their unique properties, such as biocompatibility and signal amplification, and their ability to bind
covalently to Abs via their functional groups [46,47]. In this subsection, we discuss application of MNPs to electrochemical
immunosensing.
Human epidermal growth factor receptor 2 (HER2), as a key prognostic marker [48], is over-expressed in 10–25% of breast cancers
[49], which are some of the most common malignant types of tumors
in women [50]. Establishment of a fast technique sensitive to the
low levels of the HER2 biomarker, which results in early diagnosis
of the cancer, is very important for not only increasing the survival rate, but also saving cost and time in successful prognosis of the
disease. Recently, Emami et al. [51] attached different proportions
of antiHER2 Ab to pegylated Fe3O4-MNPs to form highly-loaded
bioconjugates. To design a label-free platform, the most appropriate bioconjugate was stabilized covalently on the surface of a gold
electrode to assay ultra-low levels of HER2 antigen in serum samples.
The immunosensor proposed by this group was linearly responsive to HER2 concentrations over the ranges 0.01–10 ng/mL and
10–100 ng/mL and benefited from a satisfactory LOD as low as
0.995 pg/mL.
Also, a novel magnetic field-controllable, disposable electrochemical immunosensor for rapid determination of clenbuterol (CLB)
was fabricated by GN sheets (GS)-Nafion (Nf) film dropped on the
screen-printed carbon electrode (SPCE) and then Fe3O4-AuNPs
(GoldMag particles, GMPs) coated with bovine serum albuminCLB (BSA-CLB) conjugates were absorbed on it with the aid of an
external magnetic field [52]. In this report, the LOD was 0.22 ng/mL.
Interestingly, a novel electrochemical-sensing platform based on
magnetic field-induced self-assembly of [email protected] NPs
([email protected]) was fabricated for the sensitive detection of creatinine in biological fluids [53]. In this work, template-molecule
creatinine was self-assembled on the surface of [email protected] together with functional monomer aniline by forming N-H bonds. After
pre-assembly, through induction of the magnetic GCE (MGCE), the
ordered structure of molecularly-imprinted polymers (MIPs) was
established by electropolymerization and assembled on the surface
of the MGCE with the help of magnetic fields by a simple onestep approach. The route to fabrication of MIES is shown in Fig. 4.
From this report, it was found that the stable and hydrophilic [email protected]
PANI can not only provide available functionalized sites with which
the template molecule creatinine can form hydrogen bond by the
abundant amino groups in PANI matrix, but also promote a pathway
for electron transfer. It is important to point out that this molecularlyimprinted electrochemical sensor (MIES) shows an LOD 0.35 nmol/L
for determination of creatinine.
The simultaneous determination of numerous tumor markers
plays an important role in early screening of many cancers, and can
obtain obviously higher specificity for diagnosis than single tumormarker determination [54]. Hence, it is important to realize
simultaneous determination of multi-analytes in a single run. Recently, Wang et al. [55] prepared a novel electrochemical multiplexed
immunoassay for simultaneous determination of alpha-fetoprotein
(AFP) and carcinoembryonic antigen (CEA) using recombinant
Fig. 4. Preparation of MIES [53].
M. Hasanzadeh et al./Trends in Analytical Chemistry 72 (2015) 1–9
apoferritin-encoded metallic NPs (rApo-M) as labels and dualtemplate magnetic MIPs (MMIPs) as capture probes. In this report,
the capture probes were synthesized by self-polymerization of dopamine (DA) on the Fe3O4-MNPs using AFP and CEA as the template
proteins, which were used to enrich the targets simultaneously. According to experimental results of this report, the immunoassay
enabled simultaneous determination of AFP and CEA in a single run
with wide dynamic ranges of 0.001–5 ng/mL, and the LODs of AFP
and CEA were 0.3 pg/mL and 0.35 pg/mL, respectively.
Similar work was reported by Yang et al. [56], whose dual signalamplification strategy was proposed based on hollow-platinum NPdecorated Fe3O4-NPs (HPtNPs-Fe3O4) and glucose oxidase (GOD) for
sensitive detection of α-1-fetoprotein (AFP). The LOD was 0.22 ng/mL.
In applying MNPs to electrochemical detection of pathogens, there
is an immediate need for rapid methods to detect and to determine pathogenic bacteria in food products as alternatives to the
laborious, time-consuming culture procedures. Recently, two electrochemical immunosensors were developed for this purpose.
In the first report, Freitas et al. [57] prepared an electrochemical
immunoassay using iron-oxide/gold-core/shell NPs ([email protected]) conjugated with anti-Salmonella Abs and applied to detection of Salmonella
typhimurium. The labeled electrochemical determination of
S. typhimurium led to an LOD of 13 cells/mL which is in the same range
as the best most recently reported LODs (0.1–500 cells/mL). Also, the
results obtained by this report showed that the method developed
by Freitas and co-workers could determine the bacteria in milk at
low concentrations and is suitable for the rapid (analysis time less
than 1 h), sensitive detection of S. typhimurium in real samples.
In the second report, an electrochemical genosensor, based on a GOmodified iron-oxide-chitosan hybrid nanocomposite for pathogen
detection, was prepared by Tiwari et al. [58]. In this report, a nucleicacid sensor was fabricated via covalent immobilization of an Escherichia
coli O157:H7 (E. coli)-specific probe oligonucleotide sequence onto a GOmodified iron-oxide-chitosan hybrid nanocomposite (GIOCh) film. These
prepared GIOCh films were electrophoretically deposited onto indiumtin oxide (ITO)-coated glass substrate, used as cathode, while a parallel
platinum plate was used as counter electrode. Using electrochemical
impedance spectroscopy, it was observed that the nanobiocomposite
prepared enhanced DNA detection and could successfully detect E. coli
in the range 10−6–10−14 M with an LOD of 10 pM.
Also, in viral infection, a novel method was proposed to detect
antigens in biological samples sensitively and selectively by exploiting the combination of functional iron MNPs with an
electrochemical immunoassay [59]. In this report, the sandwichtype immune complex was constructed as a primary antibodyantigen-secondary antibody/enzyme on the surface of MNPs. The
magnetic feature of complex NPs facilitated the fast, reliable separation of immune complexes from bio-fluids. In this work, the
covalent binding of Abs to the surface of functional MNPs was evaluated by varying the time-scale of mixing at room temperature. The
magnetically-separated NPs after Ab binding were dispersed on a
watch glass and examined by fluorescence microscopy. The
maximum brightness of green spots was clearly observed for a
sample that reacted for at least 120 min (Fig. 5). Using these results,
Cheng et al. [59] confirmed that the Abs could be immobilized onto
the functional MNPs and the amount of immobilized Abs depended on the reaction time. Finally, a prepared electrochemical
immunosensor was used for detection of hepatitis B surface antigen
and α-fetoprotein as analytes. Horseradish peroxidase (HRP)labeled secondary Abs were utilized to catalyze the oxidation of
2-aminophenol to 2-aminophenoxazin-3-one in the presence of hydrogen peroxide. The electrochemically-active 2-aminophenoxazin3-one was used to quantify antigens in the samples using
electrochemical techniques.
In this sub-section, we comprehensively summarized some of
iron MNP-based electrochemical immunosensors. More importantly,
7
Fig. 5. Fluorescence image of larger magnetic particles stained by fluorescent
hepatitis B surface antibody for 120 min [59].
we discussed in detail different aspects of the immunosensors (e.g.,
type of MNPs, injection and detection techniques, labels, analytes
and the corresponding sample matrix, and sensitivity). Consequently, we discussed several outstanding properties of the immunosensors
and their research opportunities as well as the development potential and prospects. Also, we summarized examples of MNPbased electrochemical immunosensors reported in the literature,
with their advantages and limitations, and stressed their potential
for future development.
4. Conclusion and outlook
Nanotechnology offers unique opportunities for designing
ultrasensitive biosensors and bioassays. The studies described above
demonstrate the broad potential of bioconjugated NPs for amplified transduction of biomolecular recognition events. Given optical
and electrochemical applications, such NP labels provide the basis
for ultrasensitive assays of proteins and nucleic acids. The remarkable sensitivity of the new NP-based sensing protocols opens up the
possibility of detecting disease markers, biothreat agents, or infectious agents that cannot be measured by conventional methods. Such
highly-sensitive biodetection schemes could provide early detection of diseases or warning of terrorist attack. The use of NP tags
for detecting proteins is still in its infancy, but lessons learned in
ultrasensitive DNA detection provide useful starting points. The successful realization of new signal-amplification strategies requires
proper attention to non-specific adsorption issues that commonly
control the detectability of bioaffinity assays. Proper washing and
surface blocking steps should be employed to avoid amplification
of background signals (associated with non-specific adsorption of
the NP).
Due to the minority of research being on development of new
iron and iron-oxide MNP-based electrochemical biosensors,
more electrochemical techniques should be involved in this area.
With MNPs, it will be possible for magnetic biosensors to be
applied to pre-warning and real-time detection of diseases (e.g.,
cancers).
Finally, we expect that iron and iron-oxide MNP-based electrochemical biosensors will play an increasing role in electroanalytical
science in the near future. For electroanalytical applications, research is required into the development of protocols for synthesis
and functionalization of iron and iron-oxide MNPs. For analytical
and bioanalytical applications, research should focus on increasing the sensitivity and the selectivity of nanosensors and on lifetimebased detection methodologies. The fast advancement of MNP-based
electrochemical biosensors highlights their future applications in
8
M. Hasanzadeh et al./Trends in Analytical Chemistry 72 (2015) 1–9
diverse scientific fields. The major impacts appear to be in early detection of diseases, genetic mutations and biotargets. Electrochemical
biosensors based on iron and iron-oxide MNPs provide fast, simple,
sensitive detection systems for cancer that may provide robust tools
for anticancer biosensor research.
Acknowledgments
Financial support through Drug Applied Research Center, Tabriz
University of Medical Sciences, and Urmia University are greatly
acknowledged.
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