Electrochimica Acta 84 (2012) 6273

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

Review article

Electrochemical biosensors based on magnetic micro/nano particles

Yuanhong Xu, Erkang Wang
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China

a r t i c l e

i n f o

Article history:
Received 9 December 2011
Received in revised form 25 March 2012
Accepted 28 March 2012
Available online 5 April 2012
Keywords:
Magnetic particles
Biosensors
Electrochemistry
Electrochemiluminescence

a b s t r a c t
This review shows how magnetic micro/nano particles have made signicant contributions in the developments of electrochemical and Ru(bpy)3 2+ electrochemiluminescent biosensors, including immuno-,
enzyme, DNA, aptamer ones. Reports published from 2007 to November 2011 have been covered herein.
More importantly, different aspects of the biosensors such as modes of magnetic particles, detection and
ow injection techniques, analytes and the corresponding sensitivity and sample matrix, as well as several noticeably prominent characteristics have been summarized and discussed in detail. Accordingly,
research opportunities and future development trends in these areas are discussed.
2012 Elsevier Ltd. All rights reserved.

Contents
1.
2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
MPs-based EC biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
MPs used in EC immunosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1.
Iron oxide MPs used in EC immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2.
Other MPs used in EC immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
MPs used in other EC biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Detection techniques and analytes in EC biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1.
Detection techniques in EC biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.2.
Analytes in EC biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
Several noticeable strategies in MPs-based EC biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
MPs-based ECL biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
MPs-based ECL immunosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Other MPs-based ECL biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: AFP, -fetoprotein; ASV, anodic stripping voltammetry; CEA,

anti-carcinoembryonic antigen; CNTs, carbon nanotubes; CV, cyclic voltammetry;
DPV, differential pulse voltammetry; EC, electrochemistry; ECL, electrochemiluminescence; EE2, ethinylestradiol; EIS, electrochemical impedance spectroscopy; Fc,
Ferrocene; HRP, horseradish peroxidase; LSV, linear sweep voltammetry; MGNs,
magnetic graphene nanosheets; MNPs, magnetic nanoparticles; MPs, magnetic particles; NPs, nanoparticles; SWV, square wave voltammetry; OTA, ochratoxin A; PB,
Prussian blue; SPCEs, screen printed carbon electrodes; SNPs, single-nucleotide
polymorphisms; SELEX, Systematic Evolution of Ligands by Exponential Enrichment; Th, thionine; TNT, 2,4,6-trinitrotoluene.
Corresponding author. Tel.: +86 431 85262003; fax: +86 431 85689711.
E-mail address: ekwang@ciac.jl.cn (E. Wang).
0013-4686/$ see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.electacta.2012.03.147

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1. Introduction
A biosensor is an analytical device for detecting analytes that
combines biological recognition element with a physicochemical
detector component. It consists three parts: the biological recognition element, the transducer or the detector element and the
reader device [1]. There are different types of biosensors depending on different principles. According to the transducer types,
biosensors can be classied as optical, thermal, piezoelectric, electrochemistry (EC), electrochemiluminescence (ECL) biosensors, etc.

Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 6273

EC process can directly convert a biological event to an electronic

signal, including the measurable current (amperometric), potential or charge accumulation (potentiometric) or altered conductive
properties of a medium (conductometric) between electrodes [2].
The most common techniques include cyclic voltammetry (CV),
differential pulse voltammetry (DPV), square wave voltammetry
(SWV), linear sweep voltammetry (LSV), anodic stripping voltammetry (ASV) amperometry, potentiometry, electrochemical
impedance spectroscopy (EIS) and so on [2]. ECL is a means to
emit measurable luminescent signals by converting EC energy
into radiative energy via an EC reaction. ECL belongs to one special form of EC and is stated separately herein. Among these, the
ECL of tris(2,2 -bipyridyl)ruthenium(II) [Ru(bpy)3 2+ ] (including its
analogues) and its coreactants systems has received much more
considerable attention. Both EC and ECL biosensors possess advantages of rapid, high sensitivity, simple instrumentation, low cost
and ease of miniaturization, thus provide attractive means in many
areas including clinical, biological, pharmaceutical, forensic, environmental and agricultural applications, etc. [3,4].
EC and ECL immuno-, enzyme, tissue and DNA biosensors are
designed through immobilizing biological recognition elements of
antibodies, enzyme, tissue and DNA on the working electrode surface, respectively. To improve the sensitivity of biosensors, signal
amplication process is needed. With the development of microand nanotechnology, micro/nanoparticles with optical, electronic
and magnetic properties could be combined to the development of
biosensors. The micro/nanoparticles could be immobilized on the
surface of the transducers by ways of physical adsorption, chemical covalent bonding, electrodeposition and so on for EC or ECL
signal generation and amplication [5]. One main factor to evaluate an EC or ECL biosensor is reproducible regeneration of the
sensing surface. This renewal is a difcult task since it requires
renewing the recognition element bound to the transducer surface. Moreover, this drawback makes the biosensors difcult to be
integrated into automatic systems [6]. An alternative approach to
reach this renewal is applying the disposable magnetic particles
(MPs) to build up the biosensors. With the assistance of an external
magnet, the in situ biosensing surface is built up by localizing the
recognition element-coated MPs on the electrode area. The electrode surface can be easily renewed by alternate positioning of the
external magnet. Meanwhile, MPs provide a high surface area to
immobilize the biomolecules as many as possible, resulting in a
lower detection limit. Moreover, the use of MPs can play the roles
of concentration and purication. It is particularly efcient in detecting analytes in complex sample matrix, which may exhibit either
poor mass transport to biosensor or physical blockage of biosensor surface by non-specic adsorption [6]. MPs-based techniques
can remove the need for sample pretreatment by centrifugation or
chromatography, thus shortening the handling time [3]. In addition,
most of the MPs, especially the iron oxide ones, are biocompatible
and non-genotoxic; they can either be applied for simple adsorption of biomolecules, or functionalized or encapsulated in polymers
or metal or silica NPs or carbon materials to enhance the biocompatibility and increase the functionalities [7,8]. Thus, MPs have been
providing a promising experimental platform for developing both
EC and ECL biosensors.
In this early century, Sole et al. [7] summarized the analytical use
of magnetic beads as new materials for EC biosensing and presented
considerable prospective aspects of this scientic eld. As expected,
the number of publications per year on MPs related to EC and ECL
biosensors shows an increasing trend in recent years, especially
between 2009 and 2011 (see in Fig. 1A). It clearly indicated that
more and more scientists are participating into this research eld.
Except for the research works, some literature reviews have begun
to refer to these areas. For example, Guo and Dong [5] and Wang and
Hu [9] gave general reviews of recent advances before the year 2008

63

of inorganic nanoparticles (NPs) such as metal, semi-conducting,

magnetic and solid oxide, and hybrid ones for enhancing construction of EC biosensors. Grieshaber et al. [2] described the principles
and architectures of the EC biosensors, magnetic nanoparticles
(MNPs)-based biosensing was partly mentioned. Specic review
of MPs as versatile tools for EC or ECL biosensing were reported,
but attention was only particularly paid on DNA hybridization sensors [10] or immunosensors [11] or detecting biomolecules (nucleic
acids and proteins) and cells before the year 2007 [3]. Applications
of Ru(bpy)3 2+ ECL in bioanalysis was summarized by Wei and Wang
[4], but only aptamer biosensor was mentioned. So far, there has
been no recent overview on the use of MPs for developing both EC
and ECL biosensors based on various types of biological recognition
elements and for detecting a variety of analytes.
Fig. 1B shows a statistical study according to the different recognition elements used in the MPs-based EC and ECL biosensors.
Most applications of MPs are concentrated on the development of
immunosensors, followed by DNA and enzyme biosensors. Since
screened through the iterative process referred to as Systematic
Evolution of Ligands by Exponential Enrichment (SELEX) from the
combinatorial libraries of synthetic nucleic acid, aptamers-related
analytical research has experienced explosive growth over the past
few years [5]. Thus aptamer biosensor is classied as one separate
section to be discussed herein.
Based on the previous review works, combining with the
selected latest research articles from 2007 to November 2011,
various MPs-based EC and Ru(bpy)3 2+ ECL biosensors including
immuno-, enzyme, DNA and aptamer ones are comprehensively
summarized in this review. More importantly, different aspects
of the biosensors such as modes of MPs, injection and detection
techniques, labels, analytes and the corresponding sample matrix,
the sensitivity, etc. are discussed in detail. Consequently, several
outstanding properties of the biosensors and their research opportunities as well as the development potential and prospects are
discussed.

2. MPs-based EC biosensors
2.1. MPs used in EC immunosensors
Generally, EC immunosensors are based on the use of electrodes
as solid-phase and as EC transducers, antibody or antigen molecules
are directly immobilized at the sensor surface (transducer), on
which EC signal change is measured before and after the antigenantibody interaction [12]. EC immunosensors with simplicity,
portability, high selectivity and sensitivity have obtained considerable attention and evolved dramatically over the past decades
[13,14]. However, the use of the electrode surface as solid phase
and EC transducer bring with some problems and retard its practical applications: (1) biospecically bound antibody molecules
can shield the sensing surface, which results in steric hindrance
of the electron transfer, causing a reduced EC signal; (2) antibody immobilization to the electrode surface is time consuming;
(3) their reusability is limited due to the permanent immobilization of the antibody or antigen molecules on the electrode
surface [12,14]. An alternative approach to resolve this problem
is applying other solid-phase such as MPs for the separation of
biorecognition complexes and for the amplied EC sensing of antigen/antibody complexes [12,15]. Use of MPs brings with reusable
electrode surface and greatly improve the performance of the EC
biosensors because of (1) the increased binding capacity resulting
from the large surface area of MPs; (2) the faster assay kinetics
due to the MPs are in suspension and the target does not have to
migrate very far [12]. Both MPs and functionalized MPs have been
extensively applied in the development of the EC immunosensors

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Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 6273

Fig. 1. A. Number of publications, about which MPs were used in the development of EC and ECL biosensors; B. Percentages of classied publications.
Source: ISI Web of Knowledge from 2007 to November 2011.

for a broad variety of analytical and bio-analytical applications

[1273]. These MPs are usually modied by various linkage
groups for antibody attachment. Streptavidin or avidin coated MPs
would be able to capture biotin antibodies though the avidinbiotin interaction [16,21,24,26,30,33,50,54,56,64], which is with
extremely high afnity and specicity and resistant to extremes
of heat, pH and proteolysis. Antibodies can also be successfully immobilized onto MPs through amidation reaction between
amino/carboxyl functioned MPs and carboxyl/amino modied
antibodies [13,19,20,34,36,59,72]. Tosyl groups [22,28,67,68,73],
protein A [37,41,42] and protein G [12,27,39,49,63,107] terminated
MPs are also means to couple or immobilize or conjugate the antibodies to the MPs surfaces. Due to the sophisticated ways of both
synthesizing and modifying the MPs with various afnity groups,
many functionalized MPs have been commercially available now.
Commercial MPs used in these immunosensors were typically paramagnetic particles in the micro scale (15 m), which presented
polymeric coatings on the outside of the MNPs core [3,17].
2.1.1. Iron oxide MPs used in EC immunosensors
Due to the simple preparation, superparamagnetic property,
controlled similar size as the antibodies and the high specic area
for antibody attachment, iron oxide MPs (Fe2 O3 and Fe3 O4 ) are
the most commonly used ones in developing immunosensors until
now. However, because of the magnetic dipolar attraction and
their large ratio of surface area to volume, most unmodied MPs
are easily aggregated into clusters when they directly exposed to
the biological solutions. To overcome this shortcomings, also to
enhance their biocompatibility and bring new functionalities to
the MPs, a broad variety of functionalized MPs have been synthesized [3,25], such as coreshell Fe3 O4 SiO2 NPs [15,18], coreshell
Fe3 O4 gold NPs [23,25,35,36,43,48,57,58], coreshell Fe3 O4 silver
nanocomposites [53], Fe3 O4 NPs modied with O-carboxymethyl
chitosan [38], Prussian blue (PB) [44], ZrO2 [51] and so on. Among
them, coreshell Fe3 O4 SiO2 NPs is one of the most frequently
used in biosensing [15,18]. This format not only stabilized the
NPs in solution, but also favored the binding of bio-ligands (e.g.
antibodies) on the NPs surface. Moreover, the SiO2 provides an
excellent surface for further modication with specic purpose. For
example, while functionalized with epoxy groups, this coreshell
Fe3 O4 SiO2 NPs greatly increased the amount and activity of the
immobilized antibodies [18]. Due to its easy preparation, large
specic surface area, excellent biocompatibility, strong adsorption
ability and good conductivity, etc. of gold NPs, the structure of
coreshell Fe3 O4 gold NPs is a considerably more attractive platform for EC biosensing [23,25]. By further functionalization of this
structure with a PB interlayer [25], or a self-assembled monolayer of 11-mercaptoundecanoic acid [43], or layer-by-layer of

negatively charged mercaptosuccinic acid and positively charged

poly(l-lysine) [35], reproducible immunosensors with high sensitivity could be achieved for detection of antigens [25,35] and
immunological interaction between human IgG [43], etc. Thereinto,
PB, as a good electron mediator, could provide a desirable environment for the electron transfer process between the immobilized
biomolecules and the base electrode and lead to amplied signal
output [25,44]. When using a porous structure of polymer microspheres to encapsulate the MPs, magnetic polymer microspheres
were formed. Due to the large specic surface, they alleviate the difculty of separating polymer or inorganic sorbents from complex
mixtures. However, only limited amounts of magnetic compounds
can be precipitated into the porous structure, and the release of
iron oxide from the pores is even serious. To reduce these limits,
it is benecial to make the microspheres with micro- (<2 nm) or
mesopores (250 nm), in which the iron oxide can be easily kept.
For this purpose, homogeneous poly(styrene-co-divinylbenzene)
microspheres with a rather narrow size distribution were prepared
by Salek et al. [61]. The obtained microspheres were chloromethylated and then hypercrosslinked to form porous structure and
provide sufciently large space for precipitation of iron oxides.
Anti-ovalbumin was then immobilized on the surface of the
magnetic microspheres without adversely inuencing the functions of the antibodies. Finally, a sandwich-type electrochemical
immunosensor was successfully established. With the aid of other
advanced materials, new ways of MPs that used for antibody attachment appeared. For example, antibody and enzyme can be directly
xed on nanozirconium dioxide (nano-ZrO2 ) and keep the biological activity for a long time due to the large supercial area of
nano-ZrO2 . Also DNA can specially combine with ZrO2 . Thus by
using nano-ZrO2 covering to the Fe3 O4 MPs, DNA-derived magnetic nanochain probes could be formed for developing a novel
reusable sandwich EC immunosensor for separation, enrichment
and sensitive detection of -fetoprotein (AFP) in human serum [51].
Graphene nanosheet is an attractive two-dimensional nanomaterial with large specic surface area (2600 m2 g1 ) and high electron
transfer rate (15 000 cm2 V1 s1 ). By patterning biofunctionalized
MNPs assemblies onto the graphene nanosheets, a new and lter
like hybrid nanomaterial was obtained, named magnetic graphene
nanosheets (MGNs). Combined with a ow-through system, the
magnetic graphene immunosensing platform could simultaneously
immobilize two types of antibodies and efciently capture the
targets. And nally a multiplexed immunoassay method was successfully established [70].
2.1.2. Other MPs used in EC immunosensors
Meanwhile, other nanosized MPs, such as CoFe2 O4 [20,31]
and NiCo2 O4 [66,69] have been synthesized and investigated for

Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 6273

65

Fig. 2. Fabrication process and schematic illustration of the NiCo2 O4 -Nf-Th-Au nanomaterials, and measurement protocol of the EC immunosensor.
Reprinted with permission from Ref. [66] (Copyright 2011, American Chemical Society).

EC biosensing applications. For example, CoFe2 O4 NPs possess

remarkable electrical and magnetic properties [20], Tang et al. [31]
synthesized a kind of multifunctional MPs with the CoFe2 O4 NPs
as the core and PB NPs-doped silica as the shell for the attachment of biomolecules. CoFe2 O4 NPs not only provided the substrate
for the formation of the PBNPs-doped silica NPs, but also speed
the separation and purication of biomolecule-functionalized MPs
as well as the fabrication of the reusable EC immunosensors. Li
et al. [66] initially synthesized magnetic mesoporous NiCo2 O4
nanosheets with three-dimension channels, and then fabricated
the organicinorganic NiCo2 O4 -Naon-thionine-nanogold hybrid
nanomaterials on the magnetic mesoporous nanosheets (shown
in Fig. 2). This novel bionanomaterial was further explored for
the signal amplication of EC immunosensors [66,69]. It showed
good adsorption properties for the attachment of horseradish peroxidase (HRP)-labeled secondary anti-carcinoembryonic antigen
(CEA) antibody and could enhance the sensitivity and reproducibility of sensors. Through a sandwich-type immunoassay, the
biosensor allowed the detection of CEA at a concentration as low
as 0.5 pg mL1 [66].
2.2. MPs used in other EC biosensors
For other EC biosensors such as enzyme, DNA and aptamer
ones, most MPs are similar as the ones for EC immunosensors, such as streptavidin-coated [93,94,97,103,132,135,137,146],
carboxyl-modied [102,109,131,134,137] and coreshell MNPs
[79,83,8688,106,111,114,115,140] and so on. Detailed information can be found in Tables 13. Except for these, some of other
novel or functionalized MPs were also investigated to enhance the
performance of these biosensors.
For example, in developing a sensitive DNA biosensor, it was
found that magnetic microspheres coated with 4 layers polyelectrolytes could increase carboxyl groups on the surface of the
magnetic microbeads, resulting enhanced amount of capture DNA
[100]. Alginic acid-coated cobalt MPs were employed not only

for magnetic separation but also as the solid adsorbent. By being

capped with 5 -NH2 oligonucleotide, the novel MPs were successfully used for developing a DNA biosensor [112]. Magnetic nickel
NPs were rst utilized as an enzyme immobilization platform and
electrode material to construct screen-printing enzyme biosensors
for bisphenol A. It was found that nickel provided comparable or
better characteristics in terms of detection limit and sensitivity
than Fe3 O4 and gold NPs [84]. Carbon materials are the most widely
used to functionalize the MPs for EC enzyme biosensors, because
carbon materials such as carbon nanotubes (CNTs), graphene and
highly ordered mesoporous carbon, possess superior characteristics of extremely large surface area, controlled nanoscale structures,
and considerably high conductivity, as well as other merits of carbon [85]. For example, as a synergy existed between MNPs and CNTs
[89], by introducing nano Fe2 O3 [80] or Fe3 O4 [81] into the CNTs,
novel electrochemical nanostructured enzyme biosensors based
on CNTs could be constructed by magnetic assembly. The results
showed that the magnetic assembly method enhanced the density of CNTs and the amount of enzyme loaded on the electrode,
resulting in the improvement of the biosensors behaviors. Recently,
MNPs was found showing intrinsic peroxidase activity. Then by
incorporating MNPs as mimetic peroxidase and glucose oxidase in a
conductive mesoporous carbon, a novel strategy for developing an
efcient and robust EC biosensing platform was established. The
EC biosensors showed high sensitivity and simplicity for detection of H2 O2 and glucose, respectively [85]. Carbon-coated iron
NPs were novel MNPs with a layer of graphitic carbon coated on
the surface of nanoscale iron uniformly. The coated carbon could
protect iron from being oxidized in air and increase its dispersible
property and stability. They were dispersed in chitosan solution
and further used to immobilize HRP on the surface of polythionine
modied glassy carbon electrode by the cross-linking of glutaraldehyde. The obtained EC enzyme biosensors could be used for the
amperometric determination of H2 O2 with satisfactory results [82].
Magnetic graphene platforms [70] were also successfully applied
for EC aptamer biosensors for small molecules analysis [138,139].

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Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 6273

Table 1
Summary of MPs in development of EC enzyme biosensors for analyzing various analytes.
Detection techniques

Modes of MPs (diameter)

Analytes

Practical samples

Detection limit or
detected amount

Ref.

Amperometry
Amperometry
CV and amperometry
Amperometry
DPV
Amperometry
Amperometry

Coreshell Fe3 O4 @SiO2 (2025 nm)

CNTs lled with Fe2 O3 (10 nm)
CNTs/nano-Fe3 O4 composite (1030 nm)
Carbon-coated iron NPs (25 nm)
Coreshell CoFe2 O4 Au (18 3 nm)
Nickel NPs (30 nm)
Fe3 O4 NPs and oxidative enzymes co-entrapped in
mesoporous carbon (26 nm and 4.5 nm)
NH2 -coreshell MNPs
Three-layer AuFe3 O4 @SiO2 (Fe3 O4 @SiO2 : 360 nm, Au:
20 nm)
Coreshell Fe3 O4 mesoporous silica (450 nm)
CNTs-modied MNPs (MNPs: 100 nm)
Glutaraldehyde activated streptavidin-coated MPs
(500 nm)
Fe3 O4 /Au magnetic nanoparticulate (30 nm)

Glucose
H2 O2
Glucose
H2 O2
Glucose
Bisphenol A
Glucose

N/Aa
N/A
N/A
N/A
N/A
N/A
N/A

3.2
50 Nm
N/A
3.6 M
N/A
7.1 nM
19 to 36 nA mM1

[79]
[80]
[81]
[82]
[83]
[84]
[85]

Fructosyl valine
Glucose

Human serum
Human serum

0.1 mM
0.01 mM

[86]
[87]

Phenol
Catechol
Enzyme inhibitors

Sea water
N/A
N/A

78 mA mM1
7.61 M

[88]
[89]
[90]

Dimethoate

Chinese cabbage

5.6 104 ng mL1

Amperometry
Amperometry
Amperometry
Amperometry
Amperometry
CV and DPV

[106]

Not applicable.
b
The order of their inhibitory potency: kojic acid (IC50 = 3.7 106 M, Ki = 8.6 107 M), ascorbic acid (IC50 = 1.2 105 M), benzoic acid (IC50 = 7.2 105 M, Ki = 2.0 105 M)
and azelaic acid (IC50 = 1.3 104 M, Ki = 4.2 105 M).

2.3. Detection techniques and analytes in EC biosensors

2.3.1. Detection techniques in EC biosensors
For MPs-based EC immunosensors, different kinds of
EC detection modes were used for analyte quantication
such as CV [14,21,22,25,26,28,31,43,51], EIS [18,38,43,60],
DPV
[12,20,22,36,41,42,44,50,52,53,5557,59,66,70],
ASV
SWV
[13,14,16,19,21,65,69,73],
amperometry
[15,24,40],
[17,23,27,30,34,35,37,46,54,58,6265,67,71,72],
potentiometry
[29,49], chronoamperometry [39], conductometric measurement
[48], LSV [61] and alternating current voltammetry [107]. Similar
trends were obtained for EC DNA biosensors. While for EC enzyme
and aptamer biosensors, the most frequently used ones were
only amperometry [7982,8490] and DPV [131137,140,146],
respectively (details can be found in Tables 13). Certain EC
methods could not only be used for analytes detection, but also for
characterizing the developed biosensors, such as CV, EIS and chronoamperometry, chronocoulometry, etc. [34,35,38,39,43,60,87].
This is based on the basic principles of these EC modes, for example, EIS is a device that monitors impedance change before and
after an afnity-interaction on the sensor transduction surface. It
possesses the ability to study any intrinsic material properties or
specic processes that could inuence the conductivity/resistivity
or capacitivity of an EC system. Chronocoulometry is one similar
mode as EIS that measure the interfacial properties of the sensing
surface according to the adsorption and desorption amount of the
electroactive species on the sensing surface. All in all, advanced
biosensors have been designed on the basis of the integration
of these various EC technologies and widely used in multiple
applications.
2.3.2. Analytes in EC biosensors
MPs-based EC immunosensors were applied for different
analytes with different transducer/antibodies combinations
(The detection limits or limit range of different analytes
are placed within brackets, S/N 3.), such as clinical analysis
of
IgG
(0.8 fg mL1 1.5 ng mL1 )
[15,26,44,55,72],
1
CEA (0.5 pg mL 0.5 ng mL1 ) [13,18,20,23,25,36,66], AFP
(0.5 pg mL1 0.04 ng mL1 ) [24,25,5153], prostate specic
antigen (0.1 ng mL1 and 0.5 pg mL1 ) [17,34], hepatitis B surface antigen (87 pg mL1 ) [40], HIV antigen p24 (0.05 ng mL1 )
[35], phosphorylated acetylcholinesterase (0.15 ng mL1 ) [19],
testosterone (1.7 pg mL1 ) [37], cortisol (3.5 pg mL1 ) [42], hormone prolactin (3.74 ng mL1 ) [50], etc. in human serum or

plasma, and zearalenone (0.0070.4 ng mL1 ) [27,49,63], folic acid

(5.8 ng mL1 ) [28], salmonella (0.04 and 0.108 CFU mL1 ) [30,32],
aatoxin B1 (6 pg mL1 ) [31] and M1(0.05 pg mL1 ) [39], okadaic
acid (0.38 ng mL1 ) [56], ochratoxin A (OTA) (0.94 ng mL1 ) [60],
gliadin (24.2 ng mL1 ) [68], etc. in food samples, and polychlorinated biphenyls (0.40.8 ng mL1 ) [12], polycyclic aromatic
hydrocarbons (50 pg mL1 ) [14], 2,4,6-trinitrotoluene (TNT)
(0.1 ng mL1 ) [21], etc. in environmental samples, etc. Due to
the inherent sensitivity and simplicity of the EC techniques,
the enrichment and separation effects of MPs and the specic
interaction between antibody-antigen, highly or ultra-sensitive
detection of these analytes in complex practical samples were
achieved. Moreover, the EC immunosensors were not only used in
analyte quantication, but also used for studying immunological
interaction [43] and enzyme inhibition and phosphorylation [65]
in biological uids. It can be conclude that the EC immunosensors
have involved in various scientic disciplines and been much
mature according to the large numbers of related works. With the
further development in the near future, it has great potential to
become commercial and be applied in practical applications such
as hospital or quality control bureau.
From the summarization of recent publications, research on the
development and improvement of MPs-based EC enzyme biosensors were mostly concentrated on electrode surface materials, this
is because the performance of enzyme biosensors is largely governed by the inherent characteristic of the materials used on the
sensing surface [85]. This is also the reason why in developing
enzyme biosensors, applying new MPs or functionalizing the MPs
got more attention, while enzymes were only limited in the several model ones as well as only small amount of analytes such as
glucose, H2 O2 , phenol, etc. were reported in this eld [7990,106].
Detailed information can be found in Table 1.
MPs-based EC DNA biosensors are devices that combine an
EC transducer with a DNA probe as the recognition element
on the MPs, making use of hybridization event to detect a target DNA sequence. The determination of nucleic acid fragments
from humans, animals and viruses, etc. was the key point to
solve different problems: detections of single-nucleotide polymorphisms (SNPs) [93,95,101], hepatitis B virus [96,97], Escherichia coli
[112,114], Mycobacterium tuberculosis [117] and so on (gathered in
Table 2).
As the aptamers have been popular just in the recent decade,
not so many works were carried out in the MPs-based EC
aptamer biosensors [131140,146]. Most investigations were

Table 2
Summary of MPs in development of EC DNA biosensors for different analyzing purposes.
Modes of MPs (diameter)

Analytes

Purpose to detect

Detection limit or
detected amount

Ref.

SWV

Streptavidin-coated magnetic microspheres

(0.83 m)
Streptavidin-coated MPs
Magnetic core agarose beads with (2075 m)

Mutant DNA

SNPs

21.5 amol

[93]

Protein MutS
DNA

Single-nucleotide mismatches
Discrimination of SNPs

1.0 g mL1
N/A

[94]
[95]

Oligo(dT)25 paramagnetic beads

DNA

Hepatitis B virus

0.7 ng mL1

[96]

DNA
Guanine and adenine bases

Hepatitis B virus
Label-free DNA

43.11 nM
25 fmol

[97]
[98]

mRNA
DNA

mRNA
DNA

0.68 pM
5.0 fM

[99]
[100]

LSV

Streptavidin-coated MNPs (125 and 225 nm)

Magnetic beads Dynabeads oligo (dT)25 and
Dynabeads streptavidin M-280
DNA probes modied magnetic beads
Magnetic microspheres coated with 4 layers
polyelectrolytes (2.03.0 m)
MPs bearing dT25 strands

DNA

N/A

[101]

DPV

Carboxyl-modied MPs (1.0 m)

DNAs

1.71 pM, 1.55 pM

[102]

CV and
chronoamperometry
DPV and
chronoamperometry
1.05 V versus SCE
Chronoamperometry

Streptavidin-coated paramagnetic MPs

(1.0 0.5 m)
Streptavidin-coated paramagnetic MPs
(1.0 0.5 m)
Single Magnetic Nanobeads
Streptavidin-coated paramagnetic MPs

DNA, PNA and LNA

SNPs in p53 Mutation Hotspots

and Expression of Mutant p53
in Human Cell Lines
One-pot detection of two
targets
DNA, PNA and LNA

[103]

DNA

PCR amplied samples

51, 60 and 78 pM,

respectively
0.2 nM

[104]

DNA
DNA

N/A
0.33 nM.

[105]
[108]

DPV
CV
LSV
DPV
Chronoamperometry

Carboxyl-modied MPs (1.0 m)

Streptavidin-coated MPs (1.0 m)
Fe3 O4 /PSS/PDDA/Au composites (300 nm)
Alginic acid-coated cobalt MPs (200 nm)
Fe3 O4 magnetic NPs cysteine
Fe2 O3 @Au coreshell NPs (20 5 nm)

DNA
DNA
27-mer sequence DNA
DNA
DNA
DNA

DNA
DNA sequences of Legionella
pneumophila
Sequence-specic DNA
DNA
DNA hybridization
Escherichia coli in real water
DNA recognition
Escherichia coli (E. coli)

[109]
[110]
[111]
[112]
[113]
[114]

SWV
DPV

Gold coated ferric oxide NPs

Amine-terminated MPs (1 m)

DNA
DNA

DNA hybridization processes

Tuberculosis

5.1 1017 M
20 amol
100 aM
10 cells m1
1 nM
DNA 0.01 pM E. coli
5 cfu mL1
31 pM
0.01 g mL1

SWV
Potentiostatic control
(at 0.6 V vs Ag)
Stripping
chronopotentiometry
DPV
ASV
SWV
ASV

Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 6273

Detection techniques

[115]
[117]

67

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Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 6273

Table 3
Summary of MPs in development of EC and ECL aptamer biosensors for analyzing different analytes.
Detection
techniques

Modes of MPs (Diameter)

Analytes

Practical samples

Detection limit or
detected amount

Ref.

DPV
DPV
DPV
DPV

Streptavidin coated MPs (1.05 m)

Carboxyl-coated MNPs (100 nm)
Streptavidin-coated MPs (1.05 m)
Streptavidin-coated MPs (0.94 m)

Serum and plasma

Plasma
Serum
N/A

0.45 nM
7.82 aM
5.4 102 g mL1
769 nM, 54.5 nM

[7]
[131]
[132]
[133]

DPV
DPV

N/A
Wheat samples

6.616 1013 M
0.07 0.01 ng mL1

[134]
[135]

DPV
DPV
SWV
SWV
DPV
ECL

Carboxyl-coated MNPs (100 nm)

Streptavidin magnesphere paramagnetic beads
(1.0 0.5 m)
Streptavidin-coated MPs
Streptavidin-coated MPs and carboxyl-coated MPs
MGNs
MGNs
Coreshell Fe3 O4 Au MNPs
Streptavidin-coated MPs

Thrombin
Thrombin
C Reactive Protein
Lysozyme and
thrombin
Thrombin
OTA

N/A
Wine samples
N/A
N/A
N/A
Fetal calf serum

0.06 nM
0.11 ng mL1
0.1 nM, 0.1 nM
0.1 pM, 1.5 pM
30 fM
80 pM

[136]
[137]
[138]
[139]
[140]
[141]

ASV and ECL

Streptavidin (or carboxyl)-coated MPs

Thrombin
OTA
ATP, cocaine
ATP, cocaine
Thrombin
Platelet-derived
growth factor B-chain
homodimer
Cancer cells

N/A

[142]

ECL
ECL

Carboxyl-modied MNPs (11.5 m)

Carboxyl-modied MPs

Ramos cancer cell

Thrombin

N/A
N/A

EC: 67 cells mL1

ECL: 89 cells mL1
58 cells mL1
1.0 fM

focused on a limited number of model targets such as thrombin

[131,134,136,140,146], OTA [135,137], ATP and cocaine [138,139].
Some practical samples have been begun to study and satisfactory
results have been obtained (listed in Table 3). As far as we knew,
there is no report of the MPs-based EC biosensors in really clinical
diagnostic and therapeutic applications.
2.4. Several noticeable strategies in MPs-based EC biosensors
To endow the MPs-based EC biosensors with more prominent
properties, many valuable works have been carried out by recent
researchers:
(1) Screen printed carbon electrodes (SPCEs), which have already
commercial and widely used, have the advantages of integration of electrodes, simple manipulations, low cost and low
consumption of sample. More importantly, they can be used for
one step determination then discarded [106]. Combining with
MPs, SPCEs provided with disposable magnetic platforms in
developing inexpensive and portable EC biosensors for various
applications [19,37,41,42,50,97,106,115,132,135,137,143, etc.].
Moreover, Centi et al. [41] made the SPCEs to eight-electrode
arrays as EC transducers, which can repeat multiple analysis
and test different samples simultaneously. Using MPs as solid
phase, a disposable EC immunosensor was developed for the
detection of sulfonamides in food matrices such as honey with
ng mL1 level. The short incubation time (25 min) and the fast
EC measurement (10 s) made this proposed biosensor a possible
alternative to classic ELISA tests.
(2) In most cases, only one target analyte can be detected in one EC
cycle. Tang et al. proposed multiplexed sensing strategy by coupling the MGNs platform with distinguishable signal tags for
simultaneous electrochemical determination of CEA and AFP
[70], ATP and cocaine [139] recently. As can be seen in Fig. 3,
due to the strong noncovalent binding of MGNs with nucleobases and aromatic compounds, Ferrocene (Fc)-ATP aptamer
and thionine (Th)-cocaine aptamer were initially bound onto
the surface of MGNs. With an external magnet, the MGNs were
attracted to the surface, which activates the electrical contact
between the immobilized aptamers and the electrode, and the
sensors circuit was switched on. Fc and Th tags exhibited two
strongly well-resolved voltammetric peaks at different potentials, respectively. In the presence of the target analytes of ATP

[128]
[129]

and cocaine, the aptamers reacted with their corresponding

analytes. The specic interactions made the tagged-aptamers
release from the MGNs. Moreover, the released target-aptamer
complexes could be cleaved by the DNase I, then the targets
became free and recombined with other aptamers on the MGNs.
Cycle by cycle, it led to successive release of tagged-aptamer
from the MGNs as well as signicant decrease of the EC signal
of the functional MGNs probes. Then the targets could be determined simultaneously according to the EC signal decrease at
various peak potentials. While removed the magnet away from
the electrode, the EC behavior cycle of the functional MGNs was
switched off.
(3) In developing MPs-based EC biosensors, usually enzyme labels
and electroactive tags were applied. It should be noticed
that these modication techniques may encounter the risk
of contamination and mechanical damage of the biological
probe. Thus, some label-free techniques based on certain EC
modes such as potentiometric [29] and EIS [38] were developed to monitor the antibody-antigen interactions in real
time, without changing their properties. CEA in human serum
[29] and Campylobacter jejuni in diarrhea patients stool [38]
could be detected with the detection limit of 0.9 ng mL1
and 1.0 103 CFU mL1 , respectively. Also label-free biosensor could be realized by utilizing the intrinsic EC properties
of nucleic acid constituents such as G and A nucleobases
[94,98,102,104]. For example, detection of guanine and adenine bases at picomolar levels in acid-hydrolyzed DNA could
be easily achieved by copper-enhanced label-free anodic stripping at anodically oxidized borondoped diamond electrode.
This method showed great applicability especially when combined with the magnetoseparation in practical DNA assays [98].
Moreover, based on the sandwich-type hybridization, integrating the magnetoseparation of MPs and the amplication effect
of gold NPs, through the oxidation of purine nucleobases, a
label-free and multiplexed EC bio-barcode sensing strategy was
constructed for simultaneous detection of two target DNAs. The
detection limits of both DNA targets were achieved at pM level.
(4) Combining the ow injection analysis to the magnetic interaction, an automated magnetically controllable EC biosensor
could be fabricated [88]. Operation under ow conditions
improves immunocapture, enzymatic reactions and EC detection [62]. It could automatically control the incubation, washing
and measurement steps with acceptable reproducibility and

Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 6273

69

Fig. 3. Schematic illustration of multiplexed aptamer-based electrochemical assay using target-induced release of redox tag-conjugated aptamers from magnetic graphene
platform and DNase I-based catalytic recycling of the analyte (Th: thionine; Fc: ferrocene).
Reprinted with permission from Ref. [139] (Copyright 2011, American Chemical Society).

good stability [20]. Chen et al. [33] and Zhang et al. [105]
utilized a capillary to establish ow-injection immunosensor and DNA biosensor for CEA and DNA determination,
respectively. The capillary played the role of not only ow
injection, but also the microsampler and microreactor. It was
indicated that ow-injection biosensor systems had the advantage of simple instrumentation, which enabled easy signal

quantication and device miniaturization [20,31,70]. MPs offer

an additional advantage: the possibility to integrate magnetic
separation into microuidics technology. Because the analytes attached onto the MPs can be easily transported in a
microuidic system using pressure-driven ow, and the MPs
can be surface modied in multiple ways, the MPs together
with the external magnet would bring various functionalities

Fig. 4. (A) Schematic drawings showing: (A) a GRAVI-Cell instrument with 8-channel microchip tilted at 30 and closed lid comprising an array of eight magnets for magnetic
bead capture in each channel; (B) a top view of a GRAVI-Chip EC sensor with its inlet and outlet microchannel reservoirs and its electrode array; (C) a cross-section (along
axis a) of a microchannel, with bead trapping using an external magnet.
Reprinted with permission from Ref. [108] (Copyright 2010, Wiley-VCH).

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Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 6273

Fig. 5. The cysteamine-GNP biobar-code assay: (A) ECL nanoprobe preparation and (B) nanoparticle-based amplication scheme.
Reprinted with permission from Ref. [125] (Copyright 2010, American Chemical Society).

to a single microuidic device. In addition, EC biosensors

have the advantages of system minimization and ease of
operation [3]. Microuidic technology has the advantages of
high-throughout, portability, integration, and automation [59].
Thus, microuidic technology has now become a novel platform where different assay steps such as magnetic separation,
concentration and biological recognition of molecules and suitable EC transducers can be cleverly integrated to generate a
new biosensor [47,59,63,65,69]. For example, Rabas group coupled a microuidic magnetic immunosensor to a gold electrode
for the rapid (total assay time <30 min) and sensitive quantication of human serum IgG antibodies to Helicobacter pylori
[45], ethinylestradiol (EE2) in river water samples [46] and
zearalenone in feedstuffs samples [71], respectively. H. pylori
antigens [45], or anti-EE2 antibodies [46] or anti-zearalenone
antibodies [71] were immobilized on 3-aminopropyl-modied
MPs, respectively. The MPs were injected into the cross shape
microchannel devices and manipulated by an external removable magnet. Further based on the enzyme labeled and a
competitive direct immunoassay method, detection limits of
0.37 U mL1 , 0.09 ng L1 and 0.41 mg kg1 were obtained for
antibodies to H. pylori, EE2 and zearalenone, respectively,
which were all less than their corresponding ELISA procedures. Most works applied only one magnetic eld in the
microchannels to control either the immunointeraction or
detection procedure [45,46,58,62,64,71,95], but Hervas et al.

[63] proposed a creative strategy that based on a simple

double-T microchip layout channel of the double-T microchip,
both channels were used as immunological and enzymatic
reaction chambers, both zones were used with the aid of a
magnetic eld to avoid the non-specic adsorption in a very
simple and elegant way. Immunoassay for the zearalenone
in infant foods could be completed in less than 15 min and
with detection limit of 0.4 ng mL1 . Meanwhile, Marrazzas
group applied a high-throughout microchip to develop the
rapid and sensitive MPs-based DNA biosensors [104,108].
As can be seen in Fig. 4, the microuidic-based platform
GRAVI-Cell (DiagnoSwiss, Monthey, CH) was used. GRAVI-Cell
was a USB powered, portable, computer-controlled instrument (see Fig. 4A). The instrument worked with GRAVI-Chip
(Fig. 4B and C), which was the biosensor chip containing
eight polymer microchannels with integrated gold microelectrodes fabrication. Both hybridization and labeling events were
performed on streptavidin-coated MPs, which were immobilized with a biotinylated capture probe. Functionalized MPs
were introduced into the microchannel inlet of the chip and
accumulated near the electrode surface resulting from a magnetic holder. After hybridization with the complementary
sequence, the hybrid was tagged with an alkaline phosphatase.
The EC substrate for alkaline phosphatase revelation was paminophenyl phosphate. Solutions and reagents sequentially
passed through the microchannels, until enzyme substrate was

Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 6273

added for in situ signal determination. Upon readout, the magnet array was positioned away, MPs were removed with the
regeneration buffer, and the regenerated chip was ready for
further assays. This protocol has been applied to the analytical
detection of specic DNA sequences of Legionella pneumophila
with detection limit of 0.33 nM. The total analysis time was
about 16 min while eight samples can be processed in parallel.
3. MPs-based ECL biosensors
3.1. MPs-based ECL immunosensors
Typically, Ru(bpy)3 2+ ECL immunoassay uses streptavidincoated paramagnetic beads to bind with the biotinylated
antibodies, the beads act as the solid support and form sandwich
structure with the antigens and the ECL tag-labeled antibodies.
Then the sample is pumped through a ow cell. The magnetic sandwich sample is delivered to the electrode, When required electric
potential is exposed on the electrode, the light subsequently emit
in the presence of a Ru(bpy)3 2+ co-reactant (e.g. tri-n-propylamine)
and is measured in a photomultiplier tube and digitally recorded.
The sample is released from the electrode by removing the magnet. The cell is then washed and ready for the next assay. ECL
immunosensor provides a disposable, sensitive and selective platform for determining target proteins with short assay time and
simple operations. It has been extensively explored and commercially developed since last century [143].
To further improve its sensitivity, using the NPs to modify the
MPs is one shortcut, such as the coreshell Fe3 O4 gold NPs [78].
However, because ECL immunoassay has been mature and commercial, recent publications about MPs-based ECL immunosensors
were fewer. Even though some works have been reported, the
researcher paid most attention to the functionalized ECL tags
by MNPs or other advanced materials for signal amplication
[74,7678,143] or ultrasensitive assay of new analytes [75]. For
example, Zhan and Bard [74] applied liposomes (100-nm diameter) containing Ru(bpy)3 2+ as the ECL tag for a sandwich-type
immunoassay of human C-reactive protein with the detection limit
of 100 ng mL1 . Li et al. proposed an ultrasensitive ECL immunoassay of CEA with detection limit of 8.0 1015 M (1.6 pg mL1 ) by
using MNPs as the carrier of ECL tags for ECL signal amplication.
This ECL immunosensor was distinctive from the conventional ones
based on MPs, where the MPs were used as platforms for separation and only one or few luminophore molecules were attached to
the antibodies. In this protocol, multiple Ru(bpy)3 2+ species have
been conjugated to a MNPs that was corresponding to one antigen molecule. Thus signicant signal amplication was reached
[76]. Ultrasensitive detection of TNT contaminations in soil and
creek water samples was accomplished by the sandwich type ECL
immunosensors. Detection limit was 0.10 0.01 ppb and was
about 600-fold lower as compared with the most sensitive TNT
assay method in the literatures [75]. In these ECL immunosensors,
the used MPs were almost the normal streptavidin-coated ones
[7477,143], and the similar trend was also found for the DNA and
aptamer biosensors.

71

technique to establish these protocols, but it must be pointed out

that MPs was also one of crucial factors to reach these highly sensitive, simple, time-effective and reproducible determinations. For
example, telomerase activity was detected by the hybridization
of ECL gold nanoprobes to telomerase reaction products, subsequent capture by MPs, and in situ ECL signal measurement from
nanoprobes. Without the PCR amplication of telomerase reaction
products, telomerase activity from as little as 500 cultured cancer
cells in crude cell extracts was measured [121]. With a similar principle, as shown in Fig. 5, an ECL nanoprobe was fabricated based on
gold NPs that was modied with Ru(bpy)3 2+ -labeled cysteamine
to boost ECL signals and single strand DNA for target recognition.
The biotin labeled capture probe, target DNA, and cysteamine-gold
NPs conjugate were captured by MNPs and subsequently detected
by ECL method. As a result, detection limit of as low as 100 fM and
excellent selectivity for single-mismatched DNA detection even in
human serum were achieved [125]. This promising ECL DNA assays
proted not only from the amplication efciency of the gold NPs
(100-fold), but also from MPs that could selectively capture of the
biotinylated telomerase reaction products [121] or capture probe
DNA [125] according to the quick, reliable and strong (Kd = 1015 )
streptavidin-biotin binding interaction. In addition, with the assistance of MPs, the current telomerase assay could be easily extended
to the high-throughput, automatic and commercial ECL detection
platform [121].
ECL aptamer biosensor belongs to one extended branch of the EC
aptamer biosensors. Works in this eld were fewer than expected
(see details in Table 3). As far as we knew, only protein of plateletderived growth factor B-chain homodimer [141] and Ramos cells
[128,142] were referred to. It has proved that using MPs instead of
the electrode surface as solid support will provide a fast and simple
selection of target protein from complex matrix, and the immobilization and release of aptamer only need to activate or deactivate
the magnetic eld [141]. Ding et al. reported both the EC and ECL
aptamer biosensors for Ramos cells detection. Both methods utilized MPs as the separation tool and high afnity DNA aptamers
for signal recognition. The EC biosensor employed ASV technology
and using the unique gold NPs for signal amplication, which led to
a detection limit of 67 Ramos cells mL1 . Although the ECL biosensor was without any signal amplication of NPs, detection limit
was 89 Ramos cells mL1 , which was almost the same as the amplied EC ones [142]. According to this comparison results, it clearly
showed that much more superior properties of the ECL biosensors,
and it is promising that both ECL and MPs will contribute more to
the development of the aptamer biosensors.
Although publications on ECL enzyme biosensor have been
reported [91], but that on Ru(bpy)3 2+ ECL enzyme biosensors were
rare. This may be because of that the limited model enzyme can
coordinate with Ru(bpy)3 2+ ECL detection systems. It is anticipated
that this scientic eld will be enriched with the exploration of
more enzyme matching with Ru(bpy)3 2+ ECL applications, such as
glucose dehydrogenase [92].

4. Conclusions and outlook

4.1. Conclusions

3.2. Other MPs-based ECL biosensors

Works on MPs-based DNA biosensors have been recently
concentrated on amplication protocols for sensitive specic
detections of analytes such as plant viruses [118], plant
pathogenic bacteria [122], telomerase activity [119,121], point
mutation [120,123,126], genetically modied organism [124],
single-mismatched DNA [125], Hg2+ [127] and Listeria monocytogenes in food [130]. MPs used herein were just one assistant

Publications from 2007 to November 2011, which devoted to

the applications of MPs in the development of immuno-, enzyme,
DNA and aptamer EC and Ru(bpy)3 2+ ECL biosensors, were summarized in this review. The clever combination of different advanced
materials through different means could result in a variety of
functional micro/nano-scale MPs for the development of novel EC
and ECL biosensors with diverse functions. With different transducers/recognition elements combinations, MPs-based EC and ECL

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Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 6273

biosensors were applied for sensitively quantifying different analytes and analyzing afnity interactions in clinical, environmental,
food samples, genetic assays, etc. In addition, some noticeable and
superior characteristics of the MPs-based EC biosensors have been
discussed, such as inexpensive disposable electrode arrays, labelfree and multiplexed sensing strategy and the new microuidic
ow injection sensing platforms, etc. [128].

developments and improvements as well as in really food safety

control, environmental, clinical applications, etc.
Acknowledgments
This work is supported by the National Natural Science Foundation of China with the Grant No. 21190040 and 21075120 and 973
project 2009CB930100 and 2010CB933600.

4.2. Outlook
Despite such advances in this eld, there are still challenges to
explore new protocols and strategies for improving the sensitivity
and practical applications of the EC and ECL biosensors.
(1) With emergence of new advanced materials, such as graphene
and carbon quantum dot, the MPs will be endowed with
much more functionalities, which will probably result in more
novel MPs-based EC and ECL biosensors. In addition, although
Ru(bpy)3 2+ complex-based coreshell magnetic silica (or gold)
nanocomposites have been much developed for ECL sensors
[116], attention was mostly paid on the construction of the
sensors, analytes were usually the model tri-n-propylamine.
These nanocomposites should exert more affects on the ECL
biosensors.
(2) As a newly developed recognition element, aptamer, targets
should not be limited on the model molecules such as thrombin,
ATP and cocaine. Cells [128,142], biological toxins [135,137],
tissues and organisms are nowadays with greater research
value. These will be much more developed by virtue of SELEX
techniques. Also more EC and ECL techniques should involve
in this area. Together with the MPs, applications of aptamer
biosensors will pay more attention to the pre-warning and realtime detection of diseases such as cancers, diabetes, etc.
(3) About the injection ow system, capillary as well as capillary
electrophoresis (CE) were reported fewer, this may because
ow cells that possess good compatibility with the capillary
were fewer explored. In addition, the separation functions
of CE and microuidic electrophoresis were seldom considered. Easily understood, if taking full advantage of the CE and
microuidic electrophoresis, it will greatly enhance the separation efciency of the biosensors, thus multiplex sensing
strategy will be more easily achieved for simultaneous detection of a variety of analytes, not only two ones as mentioned in
the references [70,102,139].
(4) The noticeable characteristics of EC biosensor such as disposable electrode array, label-free, multiplex analysis and
microuidic ow injection were rarely mentioned for MPsbased ECL biosensors until now. These nicely indicate the great
opportunities for researchers in these scientic disciplines. The
intrinsic EC properties of nucleic acid constituents will provide
excellent theory support for the development of label-free ECL
DNA and aptamer biosensors. As the Ru(bpy)3 2+ immobilization
has been successfully introduced into the microchips [144,145].
It is predicted that the microuidics combing with multiple
channels and disposable array electrodes will provide powerful
platform for in-chip manipulation of MPs and ECL biosensing, and endue the biosensors with superiorities of low cost,
high sensitivity, multiplex analysis, high-throughout, integration, automation and so on.
All in all, with the increasing performance requirements of EC
and ECL biosensors, plus the emergence of new advanced materials, biological recognition elements (e.g. aptamers), injection and
separation techniques with innovative and superior properties,
MPs will certainly offer greater benets for EC and ECL biosensors

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