R

E

YS

a

ARRAA

KMBEE

C

aDnFmtPpm

0h

Electrochimica Acta 84 (2012) 62– 73

Contents lists available at SciVerse ScienceDirect

Electrochimica Acta

j ourna l ho me pag e: www.elsev ier .com/ locate /e lec tac ta

eview article

lectrochemical biosensors based on magnetic micro/nano particles

uanhong Xu, Erkang Wang ∗

tate Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China

r t i c l e i n f o

rticle history:eceived 9 December 2011eceived in revised form 25 March 2012ccepted 28 March 2012

a b s t r a c t

This review shows how magnetic micro/nano particles have made significant contributions in the devel-opments 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

vailable online 5 April 2012

eywords:agnetic particles

iosensorslectrochemistry

flow injection techniques, analytes and the corresponding sensitivity and sample matrix, as well as sev-eral 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.

lectrochemiluminescence

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622. MPs-based EC biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

2.1. MPs used in EC immunosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632.1.1. Iron oxide MPs used in EC immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642.1.2. Other MPs used in EC immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

2.2. MPs used in other EC biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652.3. Detection techniques and analytes in EC biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

2.3.1. Detection techniques in EC biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662.3.2. Analytes in EC biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

2.4. Several noticeable strategies in MPs-based EC biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683. MPs-based ECL biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

3.1. MPs-based ECL immunosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713.2. Other MPs-based ECL biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

4. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714.1. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714.2. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Abbreviations: AFP, �-fetoprotein; ASV, anodic stripping voltammetry; CEA,nti-carcinoembryonic antigen; CNTs, carbon nanotubes; CV, cyclic voltammetry;PV, differential pulse voltammetry; EC, electrochemistry; ECL, electrochemilumi-escence; EE2, ethinylestradiol; EIS, electrochemical impedance spectroscopy; Fc,errocene; HRP, horseradish peroxidase; LSV, linear sweep voltammetry; MGNs,agnetic graphene nanosheets; MNPs, magnetic nanoparticles; MPs, magnetic par-

icles; NPs, nanoparticles; SWV, square wave voltammetry; OTA, ochratoxin A; PB,russian blue; SPCEs, screen printed carbon electrodes; SNPs, single-nucleotideolymorphisms; SELEX, Systematic Evolution of Ligands by Exponential Enrich-ent; 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).

013-4686/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.electacta.2012.03.147

1. Introduction

A biosensor is an analytical device for detecting analytes thatcombines biological recognition element with a physicochemicaldetector component. It consists three parts: the biological recog-nition element, the transducer or the detector element and thereader device [1]. There are different types of biosensors depend-

ing on different principles. According to the transducer types,biosensors can be classified as optical, thermal, piezoelectric, elec-trochemistry (EC), electrochemiluminescence (ECL) biosensors, etc.

himic

EstpTd(tieicEactaar

dafaaabsisusrfir(mrteilotpsccmatoppE

octbbmEtH

Y. Xu, E. Wang / Electroc

C process can directly convert a biological event to an electronicignal, including the measurable current (amperometric), poten-ial or charge accumulation (potentiometric) or altered conductiveroperties of a medium (conductometric) between electrodes [2].he most common techniques include cyclic voltammetry (CV),ifferential pulse voltammetry (DPV), square wave voltammetrySWV), linear sweep voltammetry (LSV), anodic stripping vol-ammetry (ASV) amperometry, potentiometry, electrochemicalmpedance spectroscopy (EIS) and so on [2]. ECL is a means tomit measurable luminescent signals by converting EC energynto radiative energy via an EC reaction. ECL belongs to one spe-ial form of EC and is stated separately herein. Among these, theCL of tris(2,2′-bipyridyl)ruthenium(II) [Ru(bpy)3

2+] (including itsnalogues) and its coreactants systems has received much moreonsiderable attention. Both EC and ECL biosensors possess advan-ages of rapid, high sensitivity, simple instrumentation, low costnd ease of miniaturization, thus provide attractive means in manyreas including clinical, biological, pharmaceutical, forensic, envi-onmental and agricultural applications, etc. [3,4].

EC and ECL immuno-, enzyme, tissue and DNA biosensors areesigned through immobilizing biological recognition elements ofntibodies, enzyme, tissue and DNA on the working electrode sur-ace, respectively. To improve the sensitivity of biosensors, signalmplification process is needed. With the development of micro-nd nanotechnology, micro/nanoparticles with optical, electronicnd magnetic properties could be combined to the development ofiosensors. The micro/nanoparticles could be immobilized on theurface of the transducers by ways of physical adsorption, chem-cal covalent bonding, electrodeposition and so on for EC or ECLignal generation and amplification [5]. One main factor to eval-ate an EC or ECL biosensor is reproducible regeneration of theensing surface. This renewal is a difficult task since it requiresenewing the recognition element bound to the transducer sur-ace. Moreover, this drawback makes the biosensors difficult to bentegrated into automatic systems [6]. An alternative approach toeach this renewal is applying the disposable magnetic particlesMPs) to build up the biosensors. With the assistance of an external

agnet, the in situ biosensing surface is built up by localizing theecognition element-coated MPs on the electrode area. The elec-rode surface can be easily renewed by alternate positioning of thexternal magnet. Meanwhile, MPs provide a high surface area tommobilize the biomolecules as many as possible, resulting in aower detection limit. Moreover, the use of MPs can play the rolesf concentration and purification. It is particularly efficient in detec-ing analytes in complex sample matrix, which may exhibit eitheroor mass transport to biosensor or physical blockage of biosen-or surface by non-specific adsorption [6]. MPs-based techniquesan remove the need for sample pretreatment by centrifugation orhromatography, thus shortening the handling time [3]. In addition,ost of the MPs, especially the iron oxide ones, are biocompatible

nd non-genotoxic; they can either be applied for simple adsorp-ion of biomolecules, or functionalized or encapsulated in polymersr metal or silica NPs or carbon materials to enhance the biocom-atibility and increase the functionalities [7,8]. Thus, MPs have beenroviding a promising experimental platform for developing bothC and ECL biosensors.

In this early century, Sole et al. [7] summarized the analytical usef magnetic beads as new materials for EC biosensing and presentedonsiderable prospective aspects of this scientific field. As expected,he number of publications per year on MPs related to EC and ECLiosensors shows an increasing trend in recent years, especiallyetween 2009 and 2011 (see in Fig. 1A). It clearly indicated that

ore and more scientists are participating into this research field.

xcept for the research works, some literature reviews have beguno refer to these areas. For example, Guo and Dong [5] and Wang andu [9] gave general reviews of recent advances before the year 2008

a Acta 84 (2012) 62– 73 63

of inorganic nanoparticles (NPs) such as metal, semi-conducting,magnetic and solid oxide, and hybrid ones for enhancing construc-tion of EC biosensors. Grieshaber et al. [2] described the principlesand architectures of the EC biosensors, magnetic nanoparticles(MNPs)-based biosensing was partly mentioned. Specific reviewof MPs as versatile tools for EC or ECL biosensing were reported,but attention was only particularly paid on DNA hybridization sen-sors [10] or immunosensors [11] or detecting biomolecules (nucleicacids and proteins) and cells before the year 2007 [3]. Applicationsof Ru(bpy)3

2+ ECL in bioanalysis was summarized by Wei and Wang[4], but only aptamer biosensor was mentioned. So far, there hasbeen no recent overview on the use of MPs for developing both ECand ECL biosensors based on various types of biological recognitionelements and for detecting a variety of analytes.

Fig. 1B shows a statistical study according to the different recog-nition elements used in the MPs-based EC and ECL biosensors.Most applications of MPs are concentrated on the development ofimmunosensors, followed by DNA and enzyme biosensors. Sincescreened through the iterative process referred to as SystematicEvolution of Ligands by Exponential Enrichment (SELEX) from thecombinatorial libraries of synthetic nucleic acid, aptamers-relatedanalytical research has experienced explosive growth over the pastfew years [5]. Thus aptamer biosensor is classified as one separatesection to be discussed herein.

Based on the previous review works, combining with theselected latest research articles from 2007 to November 2011,various MPs-based EC and Ru(bpy)3

2+ ECL biosensors includingimmuno-, enzyme, DNA and aptamer ones are comprehensivelysummarized in this review. More importantly, different aspectsof the biosensors such as modes of MPs, injection and detectiontechniques, labels, analytes and the corresponding sample matrix,the sensitivity, etc. are discussed in detail. Consequently, severaloutstanding properties of the biosensors and their research oppor-tunities as well as the development potential and prospects arediscussed.

2. MPs-based EC biosensors

2.1. MPs used in EC immunosensors

Generally, EC immunosensors are based on the use of electrodesas solid-phase and as EC transducers, antibody or antigen moleculesare directly immobilized at the sensor surface (transducer), onwhich EC signal change is measured before and after the antigen-antibody interaction [12]. EC immunosensors with simplicity,portability, high selectivity and sensitivity have obtained consid-erable attention and evolved dramatically over the past decades[13,14]. However, the use of the electrode surface as solid phaseand EC transducer bring with some problems and retard its prac-tical applications: (1) biospecifically bound antibody moleculescan shield the sensing surface, which results in steric hindranceof the electron transfer, causing a reduced EC signal; (2) anti-body immobilization to the electrode surface is time consuming;(3) their reusability is limited due to the permanent immobi-lization of the antibody or antigen molecules on the electrodesurface [12,14]. An alternative approach to resolve this problemis applying other solid-phase such as MPs for the separation ofbiorecognition complexes and for the amplified EC sensing of anti-gen/antibody complexes [12,15]. Use of MPs brings with reusableelectrode surface and greatly improve the performance of the ECbiosensors because of (1) the increased binding capacity resulting

from the large surface area of MPs; (2) the faster assay kineticsdue to the MPs are in suspension and the target does not have tomigrate very far [12]. Both MPs and functionalized MPs have beenextensively applied in the development of the EC immunosensors

64 Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 62– 73

velopmS

f[gwbeofaapMbsmCmp

2

cftntatetsFnctuNaeeFisacfsl

Fig. 1. A. Number of publications, about which MPs were used in the deource: ISI Web of Knowledge from 2007 to November 2011.

or a broad variety of analytical and bio-analytical applications12–73]. These MPs are usually modified by various linkageroups for antibody attachment. Streptavidin or avidin coated MPsould be able to capture biotin antibodies though the avidin-

iotin interaction [16,21,24,26,30,33,50,54,56,64], which is withxtremely high affinity and specificity and resistant to extremesf heat, pH and proteolysis. Antibodies can also be success-ully immobilized onto MPs through amidation reaction betweenmino/carboxyl functioned MPs and carboxyl/amino modifiedntibodies [13,19,20,34,36,59,72]. Tosyl groups [22,28,67,68,73],rotein A [37,41,42] and protein G [12,27,39,49,63,107] terminatedPs are also means to couple or immobilize or conjugate the anti-

odies to the MPs surfaces. Due to the sophisticated ways of bothynthesizing and modifying the MPs with various affinity groups,any functionalized MPs have been commercially available now.

ommercial MPs used in these immunosensors were typically para-agnetic particles in the micro scale (1–5 �m), which presented

olymeric coatings on the outside of the MNPs core [3,17].

.1.1. Iron oxide MPs used in EC immunosensorsDue to the simple preparation, superparamagnetic property,

ontrolled similar size as the antibodies and the high specific areaor antibody attachment, iron oxide MPs (Fe2O3 and Fe3O4) arehe most commonly used ones in developing immunosensors untilow. However, because of the magnetic dipolar attraction andheir large ratio of surface area to volume, most unmodified MPsre easily aggregated into clusters when they directly exposed tohe biological solutions. To overcome this shortcomings, also tonhance their biocompatibility and bring new functionalities tohe MPs, a broad variety of functionalized MPs have been synthe-ized [3,25], such as core–shell Fe3O4–SiO2 NPs [15,18], core–shelle3O4–gold NPs [23,25,35,36,43,48,57,58], core–shell Fe3O4–silveranocomposites [53], Fe3O4 NPs modified with O-carboxymethylhitosan [38], Prussian blue (PB) [44], ZrO2 [51] and so on. Amonghem, core–shell Fe3O4–SiO2 NPs is one of the most frequentlysed in biosensing [15,18]. This format not only stabilized thePs in solution, but also favored the binding of bio-ligands (e.g.ntibodies) on the NPs surface. Moreover, the SiO2 provides anxcellent surface for further modification with specific purpose. Forxample, while functionalized with epoxy groups, this core–shelle3O4–SiO2 NPs greatly increased the amount and activity of themmobilized antibodies [18]. Due to its easy preparation, largepecific surface area, excellent biocompatibility, strong adsorptionbility and good conductivity, etc. of gold NPs, the structure of

ore–shell Fe3O4–gold NPs is a considerably more attractive plat-orm for EC biosensing [23,25]. By further functionalization of thistructure with a PB interlayer [25], or a self-assembled mono-ayer of 11-mercaptoundecanoic acid [43], or layer-by-layer of

ent of EC and ECL biosensors; B. Percentages of classified publications.

negatively charged mercaptosuccinic acid and positively chargedpoly(l-lysine) [35], reproducible immunosensors with high sen-sitivity could be achieved for detection of antigens [25,35] andimmunological interaction between human IgG [43], etc. Thereinto,PB, as a good electron mediator, could provide a desirable environ-ment for the electron transfer process between the immobilizedbiomolecules and the base electrode and lead to amplified signaloutput [25,44]. When using a porous structure of polymer micro-spheres to encapsulate the MPs, magnetic polymer microsphereswere formed. Due to the large specific surface, they alleviate the dif-ficulty of separating polymer or inorganic sorbents from complexmixtures. However, only limited amounts of magnetic compoundscan be precipitated into the porous structure, and the release ofiron oxide from the pores is even serious. To reduce these limits,it is beneficial to make the microspheres with micro- (<2 nm) ormesopores (2–50 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 preparedby Salek et al. [61]. The obtained microspheres were chloromethy-lated and then hypercrosslinked to form porous structure andprovide sufficiently large space for precipitation of iron oxides.Anti-ovalbumin was then immobilized on the surface of themagnetic microspheres without adversely influencing the func-tions of the antibodies. Finally, a sandwich-type electrochemicalimmunosensor was successfully established. With the aid of otheradvanced materials, new ways of MPs that used for antibody attach-ment appeared. For example, antibody and enzyme can be directlyfixed on nanozirconium dioxide (nano-ZrO2) and keep the biolog-ical activity for a long time due to the large superficial area ofnano-ZrO2. Also DNA can specially combine with ZrO2. Thus byusing nano-ZrO2 covering to the Fe3O4 MPs, DNA-derived mag-netic nanochain probes could be formed for developing a novelreusable sandwich EC immunosensor for separation, enrichmentand sensitive detection of �-fetoprotein (AFP) in human serum [51].Graphene nanosheet is an attractive two-dimensional nanomate-rial with large specific surface area (2600 m2 g−1) and high electrontransfer rate (15 000 cm2 V−1 s−1). By patterning biofunctionalizedMNPs assemblies onto the graphene nanosheets, a new and filterlike hybrid nanomaterial was obtained, named magnetic graphenenanosheets (MGNs). Combined with a flow-through system, themagnetic graphene immunosensing platform could simultaneouslyimmobilize two types of antibodies and efficiently capture thetargets. And finally a multiplexed immunoassay method was suc-cessfully established [70].

2.1.2. Other MPs used in EC immunosensorsMeanwhile, other nanosized MPs, such as CoFe2O4 [20,31]

and NiCo2O4 [66,69] have been synthesized and investigated for

Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 62– 73 65

f-Th-AR ety).

Ersamftaentnitgo(dba

2

osc[tnp

fem[

Fig. 2. Fabrication process and schematic illustration of the NiCo2O4-Neprinted with permission from Ref. [66] (Copyright 2011, American Chemical Soci

C biosensing applications. For example, CoFe2O4 NPs possessemarkable electrical and magnetic properties [20], Tang et al. [31]ynthesized a kind of multifunctional MPs with the CoFe2O4 NPss the core and PB NPs-doped silica as the shell for the attach-ent of biomolecules. CoFe2O4 NPs not only provided the substrate

or the formation of the PBNPs-doped silica NPs, but also speedhe separation and purification of biomolecule-functionalized MPss well as the fabrication of the reusable EC immunosensors. Lit al. [66] initially synthesized magnetic mesoporous NiCo2O4anosheets with three-dimension channels, and then fabricatedhe organic–inorganic NiCo2O4-Nafion-thionine-nanogold hybridanomaterials on the magnetic mesoporous nanosheets (shown

n Fig. 2). This novel bionanomaterial was further explored forhe signal amplification of EC immunosensors [66,69]. It showedood adsorption properties for the attachment of horseradish per-xidase (HRP)-labeled secondary anti-carcinoembryonic antigenCEA) antibody and could enhance the sensitivity and repro-ucibility of sensors. Through a sandwich-type immunoassay, theiosensor allowed the detection of CEA at a concentration as lows 0.5 pg mL−1 [66].

.2. MPs used in other EC biosensors

For other EC biosensors such as enzyme, DNA and aptamernes, most MPs are similar as the ones for EC immunosen-ors, such as streptavidin-coated [93,94,97,103,132,135,137,146],arboxyl-modified [102,109,131,134,137] and core–shell MNPs79,83,86–88,106,111,114,115,140] and so on. Detailed informa-ion can be found in Tables 1–3. Except for these, some of otherovel or functionalized MPs were also investigated to enhance theerformance of these biosensors.

For example, in developing a sensitive DNA biosensor, it was

ound that magnetic microspheres coated with 4 layers poly-lectrolytes could increase carboxyl groups on the surface of theagnetic microbeads, resulting enhanced amount of capture DNA

100]. Alginic acid-coated cobalt MPs were employed not only

u nanomaterials, and measurement protocol of the EC immunosensor.

for magnetic separation but also as the solid adsorbent. By beingcapped with 5′-NH2 oligonucleotide, the novel MPs were success-fully used for developing a DNA biosensor [112]. Magnetic nickelNPs were first utilized as an enzyme immobilization platform andelectrode material to construct screen-printing enzyme biosensorsfor bisphenol A. It was found that nickel provided comparable orbetter characteristics in terms of detection limit and sensitivitythan Fe3O4 and gold NPs [84]. Carbon materials are the most widelyused to functionalize the MPs for EC enzyme biosensors, becausecarbon materials such as carbon nanotubes (CNTs), graphene andhighly ordered mesoporous carbon, possess superior characteris-tics of extremely large surface area, controlled nanoscale structures,and considerably high conductivity, as well as other merits of car-bon [85]. For example, as a synergy existed between MNPs and CNTs[89], by introducing nano Fe2O3 [80] or Fe3O4 [81] into the CNTs,novel electrochemical nanostructured enzyme biosensors basedon CNTs could be constructed by magnetic assembly. The resultsshowed that the magnetic assembly method enhanced the den-sity 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 byincorporating MNPs as mimetic peroxidase and glucose oxidase in aconductive mesoporous carbon, a novel strategy for developing anefficient and robust EC biosensing platform was established. TheEC biosensors showed high sensitivity and simplicity for detec-tion of H2O2 and glucose, respectively [85]. Carbon-coated ironNPs were novel MNPs with a layer of graphitic carbon coated onthe surface of nanoscale iron uniformly. The coated carbon couldprotect iron from being oxidized in air and increase its dispersibleproperty and stability. They were dispersed in chitosan solutionand further used to immobilize HRP on the surface of polythioninemodified glassy carbon electrode by the cross-linking of glutaralde-

hyde. The obtained EC enzyme biosensors could be used for theamperometric determination of H2O2 with satisfactory results [82].Magnetic graphene platforms [70] were also successfully appliedfor EC aptamer biosensors for small molecules analysis [138,139].

66 Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 62– 73

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

Detection techniques Modes of MPs (diameter) Analytes Practical samples Detection limit ordetected amount

Ref.

Amperometry Core–shell Fe3O4@SiO2 (20–25 nm) Glucose N/Aa 3.2 �М [79]Amperometry CNTs filled with Fe2O3 (10 nm) H2O2 N/A 50 Nm [80]CV and amperometry CNTs/nano-Fe3O4 composite (10–30 nm) Glucose N/A N/A [81]Amperometry Carbon-coated iron NPs (25 nm) H2O2 N/A 3.6 �M [82]DPV Core–shell CoFe2O4–Au (18 ± 3 nm) Glucose N/A N/A [83]Amperometry Nickel NPs (30 nm) Bisphenol A N/A 7.1 nM [84]Amperometry Fe3O4 NPs and oxidative enzymes co-entrapped in

mesoporous carbon (26 nm and 4.5 nm)Glucose N/A 19 to 36 nA mM−1 [85]

Amperometry NH2-core–shell MNPs Fructosyl valine Human serum 0.1 mM [86]Amperometry Three-layer Au–Fe3O4@SiO2 (Fe3O4@SiO2: 360 nm, Au:

20 nm)Glucose Human serum 0.01 mM [87]

Amperometry Core–shell Fe3O4–mesoporous silica (450 nm) Phenol Sea water 78 mA mM−1 [88]Amperometry CNTs-modified MNPs (MNPs: 100 nm) Catechol N/A 7.61 �M [89]Amperometry Glutaraldehyde activated streptavidin-coated MPs

(500 nm)Enzyme inhibitors N/A b [90]

CV and DPV Fe3O4/Au magnetic nanoparticulate (30 nm) Dimethoate Chinese cabbage 5.6 × 10−4 ng mL−1 [106]

a Not applicable.07 M),

a

2

2

EsD[[[[taormcnTpapsomseboa

2

a(ayC(af[tm

b The order of their inhibitory potency: kojic acid (IC50 = 3.7 × 106 M, Ki = 8.6 × 1nd azelaic acid (IC50 = 1.3 × 104 M, Ki = 4.2 × 105 M).

.3. Detection techniques and analytes in EC biosensors

.3.1. Detection techniques in EC biosensorsFor MPs-based EC immunosensors, different kinds of

C detection modes were used for analyte quantificationuch as CV [14,21,22,25,26,28,31,43,51], EIS [18,38,43,60],PV [12,20,22,36,41,42,44,50,52,53,55–57,59,66,70], ASV

15,24,40], SWV [13,14,16,19,21,65,69,73], amperometry17,23,27,30,34,35,37,46,54,58,62–65,67,71,72], potentiometry29,49], chronoamperometry [39], conductometric measurement48], LSV [61] and alternating current voltammetry [107]. Similarrends were obtained for EC DNA biosensors. While for EC enzymend aptamer biosensors, the most frequently used ones werenly amperometry [79–82,84–90] and DPV [131–137,140,146],espectively (details can be found in Tables 1–3). Certain ECethods could not only be used for analytes detection, but also for

haracterizing the developed biosensors, such as CV, EIS and chro-oamperometry, chronocoulometry, etc. [34,35,38,39,43,60,87].his is based on the basic principles of these EC modes, for exam-le, EIS is a device that monitors impedance change before andfter an affinity-interaction on the sensor transduction surface. Itossesses the ability to study any intrinsic material properties orpecific processes that could influence the conductivity/resistivityr capacitivity of an EC system. Chronocoulometry is one similarode as EIS that measure the interfacial properties of the sensing

urface according to the adsorption and desorption amount of thelectroactive species on the sensing surface. All in all, advancediosensors have been designed on the basis of the integrationf these various EC technologies and widely used in multiplepplications.

.3.2. Analytes in EC biosensorsMPs-based EC immunosensors were applied for different

nalytes with different transducer/antibodies combinationsThe detection limits or limit range of different analytesre placed within brackets, S/N ≥ 3.), such as clinical anal-sis of IgG (0.8 fg mL−1 ∼ 1.5 ng mL−1) [15,26,44,55,72],EA (0.5 pg mL−1 ∼ 0.5 ng mL−1) [13,18,20,23,25,36,66], AFP0.5 pg mL−1 ∼ 0.04 ng mL−1) [24,25,51–53], prostate specificntigen (0.1 ng mL−1 and 0.5 pg mL−1) [17,34], hepatitis B sur-

ace antigen (87 pg mL−1) [40], HIV antigen p24 (0.05 ng mL−1)35], phosphorylated acetylcholinesterase (0.15 ng mL−1) [19],estosterone (1.7 pg mL−1) [37], cortisol (3.5 pg mL−1) [42], hor-

one prolactin (3.74 ng mL−1) [50], etc. in human serum or

ascorbic acid (IC50 = 1.2 × 105 M), benzoic acid (IC50 = 7.2 × 105 M, Ki = 2.0 × 105 M)

plasma, and zearalenone (0.007–0.4 ng mL−1) [27,49,63], folic acid(5.8 ng mL−1) [28], salmonella (0.04 and 0.108 CFU mL−1) [30,32],aflatoxin B1 (6 pg mL−1) [31] and M1(0.05 pg mL−1) [39], okadaicacid (0.38 ng mL−1) [56], ochratoxin A (OTA) (0.94 ng mL−1) [60],gliadin (24.2 ng mL−1) [68], etc. in food samples, and polychlo-rinated biphenyls (0.4–0.8 ng mL−1) [12], polycyclic aromatichydrocarbons (50 pg mL−1) [14], 2,4,6-trinitrotoluene (TNT)(0.1 ng mL−1) [21], etc. in environmental samples, etc. Due tothe inherent sensitivity and simplicity of the EC techniques,the enrichment and separation effects of MPs and the specificinteraction between antibody-antigen, highly or ultra-sensitivedetection of these analytes in complex practical samples wereachieved. Moreover, the EC immunosensors were not only used inanalyte quantification, but also used for studying immunologicalinteraction [43] and enzyme inhibition and phosphorylation [65]in biological fluids. It can be conclude that the EC immunosensorshave involved in various scientific disciplines and been muchmature according to the large numbers of related works. With thefurther development in the near future, it has great potential tobecome commercial and be applied in practical applications suchas hospital or quality control bureau.

From the summarization of recent publications, research on thedevelopment and improvement of MPs-based EC enzyme biosen-sors were mostly concentrated on electrode surface materials, thisis because the performance of enzyme biosensors is largely gov-erned by the inherent characteristic of the materials used on thesensing surface [85]. This is also the reason why in developingenzyme biosensors, applying new MPs or functionalizing the MPsgot more attention, while enzymes were only limited in the sev-eral model ones as well as only small amount of analytes such asglucose, H2O2, phenol, etc. were reported in this field [79–90,106].Detailed information can be found in Table 1.

MPs-based EC DNA biosensors are devices that combine anEC transducer with a DNA probe as the recognition elementon the MPs, making use of hybridization event to detect a tar-get DNA sequence. The determination of nucleic acid fragmentsfrom humans, animals and viruses, etc. was the key point tosolve different problems: detections of single-nucleotide polymor-phisms (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 ECaptamer biosensors [131–140,146]. Most investigations were

Y. X

u, E.

Wang

/ Electrochim

ica A

cta 84 (2012) 62– 73

67

Table 2Summary of MPs in development of EC DNA biosensors for different analyzing purposes.

Detection techniques Modes of MPs (diameter) Analytes Purpose to detect Detection limit ordetected amount

Ref.

SWV Streptavidin-coated magnetic microspheres(0.83 �m)

Mutant DNA SNPs 21.5 amol [93]

SWV Streptavidin-coated MPs Protein MutS Single-nucleotide mismatches 1.0 �g mL−1 [94]Potentiostatic control(at −0.6 V vs Ag)

Magnetic core agarose beads with (20–75 �m) DNA Discrimination of SNPs N/A [95]

Strippingchronopotentiometry

Oligo(dT)25 paramagnetic beads DNA Hepatitis B virus 0.7 ng mL−1 [96]

DPV Streptavidin-coated MNPs (125 and 225 nm) DNA Hepatitis B virus 43.11 nM [97]ASV Magnetic beads Dynabeads oligo (dT)25 and

Dynabeads streptavidin M-280Guanine and adenine bases Label-free DNA 25 fmol [98]

SWV DNA probes modified magnetic beads mRNA mRNA 0.68 pM [99]ASV Magnetic microspheres coated with 4 layers

polyelectrolytes (2.0–3.0 �m)DNA DNA 5.0 fM [100]

LSV MPs bearing dT25 strands DNA SNPs in p53 Mutation Hotspotsand Expression of Mutant p53in Human Cell Lines

N/A [101]

DPV Carboxyl-modified MPs (1.0 �m) DNAs One-pot detection of twotargets

1.71 pM, 1.55 pM [102]

CV andchronoamperometry

Streptavidin-coated paramagnetic MPs(1.0 ± 0.5 �m)

DNA, PNA and LNA DNA, PNA and LNA 51, 60 and 78 pM,respectively

[103]

DPV andchronoamperometry

Streptavidin-coated paramagnetic MPs(1.0 ± 0.5 �m)

DNA PCR amplified samples 0.2 nM [104]

1.05 V versus SCE Single Magnetic Nanobeads DNA DNA N/A [105]Chronoamperometry Streptavidin-coated paramagnetic MPs DNA DNA sequences of Legionella

pneumophila0.33 nM. [108]

DPV Carboxyl-modified MPs (1.0 �m) DNA Sequence-specific DNA 5.1 × 10−17 M [109]CV Streptavidin-coated MPs (1.0 �m) DNA DNA 20 amol [110]LSV Fe3O4/PSS/PDDA/Au composites (300 nm) 27-mer sequence DNA DNA hybridization 100 aM [111]DPV Alginic acid-coated cobalt MPs (200 nm) DNA Escherichia coli in real water 10 cells m−1 [112]Chronoamperometry Fe3O4 magnetic NPs cysteine DNA DNA recognition 1 nM [113]

Fe2O3@Au core–shell NPs (20 ± 5 nm) DNA Escherichia coli (E. coli) DNA 0.01 pM E. coli5 cfu mL−1

[114]

SWV Gold coated ferric oxide NPs DNA DNA hybridization processes 31 pM [115]DPV Amine-terminated MPs (1 �m) DNA Tuberculosis 0.01 �g mL−1 [117]

68 Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 62– 73

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

Detectiontechniques

Modes of MPs (Diameter) Analytes Practical samples Detection limit ordetected amount

Ref.

DPV Streptavidin coated MPs (1.05 �m) Thrombin Serum and plasma 0.45 nM [7]DPV Carboxyl-coated MNPs (100 nm) Thrombin Plasma 7.82 aM [131]DPV Streptavidin-coated MPs (1.05 �m) C Reactive Protein Serum 5.4 × 10−2 �g mL−1 [132]DPV Streptavidin-coated MPs (0.94 �m) Lysozyme and

thrombinN/A 769 nM, 54.5 nM [133]

DPV Carboxyl-coated MNPs (100 nm) Thrombin N/A 6.616 × 10−13 M [134]DPV Streptavidin magnesphere© paramagnetic beads

(1.0 ± 0.5 �m)OTA Wheat samples 0.07 ± 0.01 ng mL−1 [135]

DPV Streptavidin-coated MPs Thrombin N/A 0.06 nM [136]DPV Streptavidin-coated MPs and carboxyl-coated MPs OTA Wine samples 0.11 ng mL−1 [137]SWV MGNs ATP, cocaine N/A 0.1 nM, 0.1 nM [138]SWV MGNs ATP, cocaine N/A 0.1 pM, 1.5 pM [139]DPV Core–shell Fe3O4–Au MNPs Thrombin N/A 30 fM [140]ECL Streptavidin-coated MPs Platelet-derived

growth factor B-chainhomodimer

Fetal calf serum 80 pM [141]

ASV and ECL Streptavidin (or carboxyl)-coated MPs Cancer cells N/A EC: 67 cells mL−1

ECL: 89 cells mL−1[142]

amos −1

hrom

f[Srtd

2

pr

(

(

ECL Carboxyl-modified MNPs (1–1.5 �m) RECL Carboxyl-modified MPs T

ocused on a limited number of model targets such as thrombin131,134,136,140,146], OTA [135,137], ATP and cocaine [138,139].ome practical samples have been begun to study and satisfactoryesults have been obtained (listed in Table 3). As far as we knew,here is no report of the MPs-based EC biosensors in really clinicaliagnostic and therapeutic applications.

.4. Several noticeable strategies in MPs-based EC biosensors

To endow the MPs-based EC biosensors with more prominentroperties, many valuable works have been carried out by recentesearchers:

1) Screen printed carbon electrodes (SPCEs), which have alreadycommercial and widely used, have the advantages of integra-tion of electrodes, simple manipulations, low cost and lowconsumption of sample. More importantly, they can be used forone step determination then discarded [106]. Combining withMPs, SPCEs provided with disposable magnetic platforms indeveloping inexpensive and portable EC biosensors for variousapplications [19,37,41,42,50,97,106,115,132,135,137,143, etc.].Moreover, Centi et al. [41] made the SPCEs to eight-electrodearrays as EC transducers, which can repeat multiple analysisand test different samples simultaneously. Using MPs as solidphase, a disposable EC immunosensor was developed for thedetection of sulfonamides in food matrices such as honey withng mL−1 level. The short incubation time (25 min) and the fastEC measurement (10 s) made this proposed biosensor a possiblealternative to classic ELISA tests.

2) In most cases, only one target analyte can be detected in one ECcycle. Tang et al. proposed multiplexed sensing strategy by cou-pling the MGNs platform with distinguishable signal tags forsimultaneous 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 nucle-obases and aromatic compounds, Ferrocene (Fc)-ATP aptamerand thionine (Th)-cocaine aptamer were initially bound ontothe surface of MGNs. With an external magnet, the MGNs wereattracted to the surface, which activates the electrical contact

between the immobilized aptamers and the electrode, and thesensor’s circuit was switched on. Fc and Th tags exhibited twostrongly well-resolved voltammetric peaks at different poten-tials, respectively. In the presence of the target analytes of ATP

cancer cell N/A 58 cells mL [128]bin N/A 1.0 fM [129]

and cocaine, the aptamers reacted with their correspondinganalytes. The specific interactions made the tagged-aptamersrelease from the MGNs. Moreover, the released target-aptamercomplexes could be cleaved by the DNase I, then the targetsbecame free and recombined with other aptamers on the MGNs.Cycle by cycle, it led to successive release of tagged-aptamerfrom the MGNs as well as significant decrease of the EC signalof the functional MGNs probes. Then the targets could be deter-mined simultaneously according to the EC signal decrease atvarious peak potentials. While removed the magnet away fromthe electrode, the EC behavior cycle of the functional MGNs wasswitched off.

(3) In developing MPs-based EC biosensors, usually enzyme labelsand electroactive tags were applied. It should be noticedthat these modification techniques may encounter the riskof contamination and mechanical damage of the biologicalprobe. Thus, some label-free techniques based on certain ECmodes such as potentiometric [29] and EIS [38] were devel-oped to monitor the antibody-antigen interactions in realtime, 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 mL−1

and 1.0 × 103 CFU mL−1, respectively. Also label-free biosen-sor could be realized by utilizing the intrinsic EC propertiesof nucleic acid constituents such as G and A nucleobases[94,98,102,104]. For example, detection of guanine and ade-nine bases at picomolar levels in acid-hydrolyzed DNA couldbe easily achieved by copper-enhanced label-free anodic strip-ping at anodically oxidized borondoped diamond electrode.This method showed great applicability especially when com-bined with the magnetoseparation in practical DNA assays [98].Moreover, based on the sandwich-type hybridization, integrat-ing the magnetoseparation of MPs and the amplification effectof gold NPs, through the oxidation of purine nucleobases, alabel-free and multiplexed EC bio-barcode sensing strategy wasconstructed for simultaneous detection of two target DNAs. Thedetection limits of both DNA targets were achieved at pM level.

(4) Combining the flow injection analysis to the magnetic inter-action, an automated magnetically controllable EC biosensor

could be fabricated [88]. Operation under flow conditionsimproves immunocapture, enzymatic reactions and EC detec-tion [62]. It could automatically control the incubation, washingand measurement steps with acceptable reproducibility and

Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 62– 73 69

Fig. 3. Schematic illustration of multiplexed aptamer-based electrochemical assay using target-induced release of redox tag-conjugated aptamers from magnetic graphenep ocene

R ciety).

Fba

R

latform and DNase I-based catalytic recycling of the analyte (Th: thionine; Fc: ferr

eprinted with permission from Ref. [139] (Copyright 2011, American Chemical So

good stability [20]. Chen et al. [33] and Zhang et al. [105]utilized a capillary to establish flow-injection immunosen-sor and DNA biosensor for CEA and DNA determination,

respectively. The capillary played the role of not only flowinjection, but also the microsampler and microreactor. It wasindicated that flow-injection biosensor systems had the advan-tage of simple instrumentation, which enabled easy signal

ig. 4. (A) Schematic drawings showing: (A) a GRAVI-Cell instrument with 8-channel micread capture in each channel; (B) a top view of a GRAVI-Chip EC sensor with its inlet anxis a) of a microchannel, with bead trapping using an external magnet.

eprinted with permission from Ref. [108] (Copyright 2010, Wiley-VCH).

).

quantification and device miniaturization [20,31,70]. MPs offeran additional advantage: the possibility to integrate magneticseparation into microfluidics technology. Because the ana-

lytes attached onto the MPs can be easily transported in amicrofluidic system using pressure-driven flow, and the MPscan be surface modified in multiple ways, the MPs togetherwith the external magnet would bring various functionalities

ochip tilted at 30◦ and closed lid comprising an array of eight magnets for magneticd outlet microchannel reservoirs and its electrode array; (C) a cross-section (along

70 Y. Xu, E. Wang / Electrochimica Acta 84 (2012) 62– 73

robe pR ciety).

Fig. 5. The cysteamine-GNP biobar-code assay: (A) ECL nanopeprinted with permission from Ref. [125] (Copyright 2010, American Chemical So

to a single microfluidic device. In addition, EC biosensorshave the advantages of system minimization and ease ofoperation [3]. Microfluidic technology has the advantages ofhigh-throughout, portability, integration, and automation [59].Thus, microfluidic technology has now become a novel plat-form where different assay steps such as magnetic separation,concentration and biological recognition of molecules and suit-able EC transducers can be cleverly integrated to generate anew biosensor [47,59,63,65,69]. For example, Raba’s group cou-pled a microfluidic magnetic immunosensor to a gold electrodefor the rapid (total assay time <30 min) and sensitive quantifi-cation of human serum IgG antibodies to Helicobacter pylori[45], ethinylestradiol (EE2) in river water samples [46] andzearalenone in feedstuffs samples [71], respectively. H. pyloriantigens [45], or anti-EE2 antibodies [46] or anti-zearalenoneantibodies [71] were immobilized on 3-aminopropyl-modifiedMPs, respectively. The MPs were injected into the cross shapemicrochannel devices and manipulated by an external remov-able magnet. Further based on the enzyme labeled and acompetitive direct immunoassay method, detection limits of0.37 U mL−1, 0.09 ng L−1 and 0.41 mg kg−1 were obtained forantibodies to H. pylori, EE2 and zearalenone, respectively,

which were all less than their corresponding ELISA proce-dures. Most works applied only one magnetic field in themicrochannels to control either the immunointeraction ordetection procedure [45,46,58,62,64,71,95], but Hervas et al.

reparation and (B) nanoparticle-based amplification scheme.

[63] proposed a creative strategy that based on a simpledouble-T microchip layout channel of the double-T microchip,both channels were used as immunological and enzymaticreaction chambers, both zones were used with the aid of amagnetic field to avoid the non-specific adsorption in a verysimple and elegant way. Immunoassay for the zearalenonein infant foods could be completed in less than 15 min andwith detection limit of 0.4 ng mL−1. Meanwhile, Marrazza’sgroup applied a high-throughout microchip to develop therapid and sensitive MPs-based DNA biosensors [104,108].As can be seen in Fig. 4, the microfluidic-based platformGRAVI-Cell (DiagnoSwiss, Monthey, CH) was used. GRAVI-Cellwas a USB powered, portable, computer-controlled instru-ment (see Fig. 4A). The instrument worked with GRAVI-Chip(Fig. 4B and C), which was the biosensor chip containingeight polymer microchannels with integrated gold microelec-trodes fabrication. Both hybridization and labeling events wereperformed on streptavidin-coated MPs, which were immobi-lized with a biotinylated capture probe. Functionalized MPswere introduced into the microchannel inlet of the chip andaccumulated near the electrode surface resulting from a mag-netic holder. After hybridization with the complementary

sequence, the hybrid was tagged with an alkaline phosphatase.The EC substrate for alkaline phosphatase revelation was p-aminophenyl phosphate. Solutions and reagents sequentiallypassed through the microchannels, until enzyme substrate was

himic

3

3

casTwpiaTnifsc

MHmwrb[eeiosuTbttbg[ciaat[a

3

cdpmsg

Y. Xu, E. Wang / Electroc

added for in situ signal determination. Upon readout, the mag-net array was positioned away, MPs were removed with theregeneration buffer, and the regenerated chip was ready forfurther assays. This protocol has been applied to the analyticaldetection of specific DNA sequences of Legionella pneumophilawith detection limit of 0.33 nM. The total analysis time wasabout 16 min while eight samples can be processed in parallel.

. MPs-based ECL biosensors

.1. MPs-based ECL immunosensors

Typically, Ru(bpy)32+ ECL immunoassay uses streptavidin-

oated paramagnetic beads to bind with the biotinylatedntibodies, the beads act as the solid support and form sandwichtructure with the antigens and the ECL tag-labeled antibodies.hen the sample is pumped through a flow cell. The magnetic sand-ich sample is delivered to the electrode, When required electricotential is exposed on the electrode, the light subsequently emit

n the presence of a Ru(bpy)32+ co-reactant (e.g. tri-n-propylamine)

nd is measured in a photomultiplier tube and digitally recorded.he sample is released from the electrode by removing the mag-et. The cell is then washed and ready for the next assay. ECL

mmunosensor provides a disposable, sensitive and selective plat-orm for determining target proteins with short assay time andimple operations. It has been extensively explored and commer-ially developed since last century [143].

To further improve its sensitivity, using the NPs to modify thePs is one shortcut, such as the core–shell Fe3O4–gold NPs [78].owever, because ECL immunoassay has been mature and com-ercial, recent publications about MPs-based ECL immunosensorsere fewer. Even though some works have been reported, the

esearcher paid most attention to the functionalized ECL tagsy MNPs or other advanced materials for signal amplification74,76–78,143] or ultrasensitive assay of new analytes [75]. Forxample, Zhan and Bard [74] applied liposomes (∼100-nm diam-ter) containing Ru(bpy)3

2+ as the ECL tag for a sandwich-typemmunoassay of human C-reactive protein with the detection limitf 100 ng mL−1. Li et al. proposed an ultrasensitive ECL immunoas-ay of CEA with detection limit of 8.0 × 10−15 M (1.6 pg mL−1) bysing MNPs as the carrier of ECL tags for ECL signal amplification.his ECL immunosensor was distinctive from the conventional onesased on MPs, where the MPs were used as platforms for separa-ion and only one or few luminophore molecules were attached tohe antibodies. In this protocol, multiple Ru(bpy)3

2+ species haveeen conjugated to a MNPs that was corresponding to one anti-en molecule. Thus significant signal amplification was reached76]. Ultrasensitive detection of TNT contaminations in soil andreek water samples was accomplished by the sandwich type ECLmmunosensors. Detection limit was ≤0.10 ± 0.01 ppb and wasbout 600-fold lower as compared with the most sensitive TNTssay method in the literatures [75]. In these ECL immunosensors,he used MPs were almost the normal streptavidin-coated ones74–77,143], and the similar trend was also found for the DNA andptamer biosensors.

.2. Other MPs-based ECL biosensors

Works on MPs-based DNA biosensors have been recentlyoncentrated on amplification protocols for sensitive specificetections of analytes such as plant viruses [118], plant

athogenic bacteria [122], telomerase activity [119,121], pointutation [120,123,126], genetically modified organism [124],

ingle-mismatched DNA [125], Hg2+ [127] and Listeria monocyto-enes in food [130]. MPs used herein were just one assistant

a Acta 84 (2012) 62– 73 71

technique to establish these protocols, but it must be pointed outthat MPs was also one of crucial factors to reach these highly sensi-tive, simple, time-effective and reproducible determinations. Forexample, telomerase activity was detected by the hybridizationof ECL gold nanoprobes to telomerase reaction products, subse-quent capture by MPs, and in situ ECL signal measurement fromnanoprobes. Without the PCR amplification of telomerase reactionproducts, telomerase activity from as little as 500 cultured cancercells in crude cell extracts was measured [121]. With a similar prin-ciple, as shown in Fig. 5, an ECL nanoprobe was fabricated based ongold NPs that was modified with Ru(bpy)3

2+-labeled cysteamineto boost ECL signals and single strand DNA for target recognition.The biotin labeled capture probe, target DNA, and cysteamine-goldNPs conjugate were captured by MNPs and subsequently detectedby ECL method. As a result, detection limit of as low as 100 fM andexcellent selectivity for single-mismatched DNA detection even inhuman serum were achieved [125]. This promising ECL DNA assaysprofited not only from the amplification efficiency of the gold NPs(∼100-fold), but also from MPs that could selectively capture of thebiotinylated telomerase reaction products [121] or capture probeDNA [125] according to the quick, reliable and strong (Kd = 10−15)streptavidin-biotin binding interaction. In addition, with the assis-tance of MPs, the current telomerase assay could be easily extendedto the high-throughput, automatic and commercial ECL detectionplatform [121].

ECL aptamer biosensor belongs to one extended branch of the ECaptamer biosensors. Works in this field were fewer than expected(see details in Table 3). As far as we knew, only protein of platelet-derived growth factor B-chain homodimer [141] and Ramos cells[128,142] were referred to. It has proved that using MPs instead ofthe electrode surface as solid support will provide a fast and simpleselection of target protein from complex matrix, and the immobi-lization and release of aptamer only need to activate or deactivatethe magnetic field [141]. Ding et al. reported both the EC and ECLaptamer biosensors for Ramos cells detection. Both methods uti-lized MPs as the separation tool and high affinity DNA aptamersfor signal recognition. The EC biosensor employed ASV technologyand using the unique gold NPs for signal amplification, which led toa detection limit of 67 Ramos cells mL−1. Although the ECL biosen-sor was without any signal amplification of NPs, detection limitwas 89 Ramos cells mL−1, which was almost the same as the ampli-fied EC one’s [142]. According to this comparison results, it clearlyshowed that much more superior properties of the ECL biosensors,and it is promising that both ECL and MPs will contribute more tothe development of the aptamer biosensors.

Although publications on ECL enzyme biosensor have beenreported [91], but that on Ru(bpy)3

2+ ECL enzyme biosensors wererare. This may be because of that the limited model enzyme cancoordinate with Ru(bpy)3

2+ ECL detection systems. It is anticipatedthat this scientific field will be enriched with the exploration ofmore enzyme matching with Ru(bpy)3

2+ ECL applications, such asglucose dehydrogenase [92].

4. Conclusions and outlook

4.1. Conclusions

Publications from 2007 to November 2011, which devoted tothe applications of MPs in the development of immuno-, enzyme,DNA and aptamer EC and Ru(bpy)3

2+ ECL biosensors, were summa-rized in this review. The clever combination of different advanced

materials through different means could result in a variety offunctional micro/nano-scale MPs for the development of novel ECand ECL biosensors with diverse functions. With different trans-ducers/recognition elements combinations, MPs-based EC and ECL

7 himica

blfsdffl

4

ea

(

(

(

(

aasM

2 Y. Xu, E. Wang / Electroc

iosensors were applied for sensitively quantifying different ana-ytes and analyzing affinity interactions in clinical, environmental,ood samples, genetic assays, etc. In addition, some noticeable anduperior characteristics of the MPs-based EC biosensors have beeniscussed, such as inexpensive disposable electrode arrays, label-ree and multiplexed sensing strategy and the new microfluidicow injection sensing platforms, etc. [128].

.2. Outlook

Despite such advances in this field, there are still challenges toxplore new protocols and strategies for improving the sensitivitynd practical applications of the EC and ECL biosensors.

1) With emergence of new advanced materials, such as grapheneand carbon quantum dot, the MPs will be endowed withmuch more functionalities, which will probably result in morenovel MPs-based EC and ECL biosensors. In addition, althoughRu(bpy)3

2+ complex-based core–shell magnetic silica (or gold)nanocomposites have been much developed for ECL sensors[116], attention was mostly paid on the construction of thesensors, analytes were usually the model tri-n-propylamine.These nanocomposites should exert more affects on the ECLbiosensors.

2) As a newly developed recognition element, aptamer, targetsshould 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 researchvalue. These will be much more developed by virtue of SELEXtechniques. Also more EC and ECL techniques should involvein this area. Together with the MPs, applications of aptamerbiosensors will pay more attention to the pre-warning and real-time detection of diseases such as cancers, diabetes, etc.

3) About the injection flow system, capillary as well as capillaryelectrophoresis (CE) were reported fewer, this may becauseflow cells that possess good compatibility with the capillarywere fewer explored. In addition, the separation functionsof CE and microfluidic electrophoresis were seldom consid-ered. Easily understood, if taking full advantage of the CE andmicrofluidic electrophoresis, it will greatly enhance the sep-aration efficiency of the biosensors, thus multiplex sensingstrategy will be more easily achieved for simultaneous detec-tion of a variety of analytes, not only two ones as mentioned inthe references [70,102,139].

4) The noticeable characteristics of EC biosensor such as dis-posable electrode array, label-free, multiplex analysis andmicrofluidic flow injection were rarely mentioned for MPs-based ECL biosensors until now. These nicely indicate the greatopportunities for researchers in these scientific disciplines. Theintrinsic EC properties of nucleic acid constituents will provideexcellent theory support for the development of label-free ECLDNA and aptamer biosensors. As the Ru(bpy)3

2+ immobilizationhas been successfully introduced into the microchips [144,145].It is predicted that the microfluidics combing with multiplechannels and disposable array electrodes will provide powerfulplatform for in-chip manipulation of MPs and ECL biosens-ing, and endue the biosensors with superiorities of low cost,high sensitivity, multiplex analysis, high-throughout, integra-tion, automation and so on.

All in all, with the increasing performance requirements of EC

nd ECL biosensors, plus the emergence of new advanced materi-ls, biological recognition elements (e.g. aptamers), injection andeparation techniques with innovative and superior properties,Ps will certainly offer greater benefits for EC and ECL biosensors

Acta 84 (2012) 62– 73

developments and improvements as well as in really food safetycontrol, environmental, clinical applications, etc.

Acknowledgments

This work is supported by the National Natural Science Founda-tion of China with the Grant No. 21190040 and 21075120 and 973project 2009CB930100 and 2010CB933600.

References

[1] M.R. Plata, A.M. Contento, A. Rios, Sensors 10 (2010) 2511.[2] D. Grieshaber, R. MacKenzie, J. Vörös, E. Reimhult, Sensors 8 (2008) 1400.[3] I.-M. Hsing, Y. Xu, W. Zhao, Electroanalysis 19 (2007) 755.[4] H. Wei, E. Wang, Luminescence 26 (2011) 77.[5] S. Guo, S. Dong, Trends in Analytical Chemistry 28 (2009) 96.[6] N. Jaffrezic-Renault, C. Martelet, Y. Chevolot, J.-P. Cloarec, Sensors 7 (2007)

589.[7] S. Sole, A. Merkoci, S. Alegret, Trends in Analytical Chemistry 20 (2001) 102.[8] L. Stanciu, Y.-H. Won, M. Ganesana, S. Andreescu, Sensors 9 (2009) 2976.[9] F. Wang, S. Hu, Microchimica Acta 165 (2009) 1.

[10] E. Palecek, M. Fojta, Talanta 74 (2007) 276.[11] S. Centi, S. Laschi, M. Mascini, Bioanalysis 1 (2009) 1271.[12] S. Centi, S. Laschi, M. Mascini, Talanta 73 (2007) 394.[13] S. Wang, X. Zhang, X. Mao, Q. Zeng, H. Xu, Y. Lin, W. Chen, G. Liu, Nanotech-

nology 19 (2008) 435501.[14] Y.-Y. Lin, G. Liu, C.M. Wai, Y. Lin, Electrochemistry Communications 9 (2007)

1547.[15] X. Mao, J. Jiang, Y. Huang, G. Shen, R. Yu, Sensors and Actuators B 123 (2007)

198.[16] H. Wu, G. Liu, J. Wang, Y. Lin, Electrochemistry Communications 9 (2007)

1573.[17] P. Sarkar, D. Ghosh, D. Bhattacharyay, S.J. Setford, A.P.F. Turner, Electroanalysis

20 (2008) 1414.[18] J. Pan, Q. Yang, Analytical and Bioanalytical Chemistry 388 (2007) 279.[19] H. Wang, J. Wang, C. Timchalk, Y. Lin, Analytical Chemistry 80 (2008) 8477.[20] X.-H. Fu, Biochemical Engineering Journal 39 (2008) 267.[21] J. Wang, G. Liu, H. Wu, Y. Lin, Analytica Chimica Acta 610 (2008) 112.[22] T. Selvaraju, J. Das, S.W. Han, H. Yang, Biosensors and Bioelectronics 23 (2008)

932.[23] D. Tang, R. Yuan, Y. Chai, Analytical Chemistry 80 (2008) 1582.[24] C. Ding, Q. Zhang, S. Zhang, Biosensors and Bioelectronics 24 (2009) 2434.[25] Y. Zhuo, P.-X. Yuan, R. Yuan, Y.-Q. Chai, C.-L. Hong, Biomaterials 30 (2009)

2284.[26] A. de la Escosura-Muniz, M.M. da Costa, A. Merkoci, Biosensors and Bioelec-

tronics 24 (2009) 2475.[27] M. Hervas, M.A. Lopez, A. Escarpa, Analytica Chimica Acta 653 (2009) 167.[28] A. Lermo, S. Fabiano, S. Hernandez, R. Galve, M.-P. Marco, S. Alegret, M.I.

Pividori, Biosensors and Bioelectronics 24 (2009) 2057.[29] X. Fu, J. Wang, N. Li, L. Wang, L. Pu, Microchimica Acta 165 (2009) 437.[30] S. Liebana, A. Lermo, S. Campoy, J. Barbe, S. Alegret, M.I. Pividori, Analytical

Chemistry 81 (2009) 5812.[31] D. Tang, Z. Zhong, R. Niessner, D. Knopp, Analyst 134 (2009) 1554.[32] S. Liebana, A. Lermo, S. Campoy, M.P. Cortes, S. Alegret, M.I. Pividori, Biosen-

sors and Bioelectronics 25 (2009) 510.[33] J. Chen, G. Zou, X. Zhang, W. Jin, Electrochemistry Communications 11 (2009)

1457.[34] V. Mani, B.V. Chikkaveeraiah, V. Patel, J.S. Gutkind, J.F. Rusling, ACS Nano 3

(2009) 585.[35] N. Gan, J. Hou, F. Hu, L. Zheng, M. Ni, Y. Cao, Molecules 15 (2010) 5053.[36] J. Li, H. Gao, Z. Chen, X. Wei, C.F. Yang, Analytica Chimica Acta 665 (2010) 98.[37] M. Eguilaz, M. Moreno-Guzman, S. Campuzano, A. Gonzalez-Cortes, P. Yanez-

Sedeno, J.M. Pingarron, Biosensors and Bioelectronics 26 (2010) 517.[38] J. Huang, G. Yang, W. Meng, L. Wu, A. Zhu, X. Jiao, Biosensors and Bioelectronics

25 (2010) 1204.[39] N. Paniel, A. Radoi, J.-L. Marty, Sensors 10 (2010) 9439.[40] G. Shen, Yun Zhang, Analytica Chimica Acta 674 (2010) 27.[41] S. Centi, A.I. Stoica, S. Laschi, M. Mascini, Electroanalysis 22 (2010) 1881.[42] M. Moreno-Guzmian, M. Eguilaz, S. Campuzano, A. Gonzalez-Cortes, P. Yanez-

Sedeno, J.M. Pingarron, Analyst 135 (2010) 1926.[43] T.T.-H. Pham, S.J. Sim, Journal of Nanoparticle Research 12 (2010) 227.[44] D. Tang, J. Tang, B. Su, H. Chen, J. Huang, G. Chen, Microchimica Acta 171 (2010)

457.[45] S.V. Pereira, G.A. Messina, J. Raba, Journal of Chromatography B 878 (2010)

253.[46] N.A. Martinez, R.J. Schneider, G.A. Messina, J. Raba, Biosensors and Bioelec-

tronics 25 (2010) 1376.

[47] D. Tang, B. Su, J. Tang, J. Ren, G. Chen, Analytical Chemistry 82 (2010) 1527.[48] J. Zhuang, T. Cheng, L. Gao, Y. Luo, Q. Ren, D. Lu, F. Tang, X. Ren, D. Yang, J.

Feng, J. Zhu, X. Yan, Toxicon 55 (2010) 145.[49] M. Hervas, M.A. Lopez, A. Escarpa, Biosensors and Bioelectronics 25 (2010)

1755.

himic

Y. Xu, E. Wang / Electroc

[50] M.M. Guzman, A. Gonzalez-Cortes, P. Yanez-Sedeno, J.M. Pingarron, AnalyticaChimica Acta 692 (2011) 125.

[51] N. Gan, L. Jia, L. Zheng, Journal of Automated Methods & Management inChemistry (2011) 957805.

[52] L. Meng, N. Gan, T. Li, Y. Cao, F. Hu, L. Zheng, International Journal of MolecularScience 12 (2011) 362.

[53] B. Su, D. Tang, J. Tang, Q. Li, G. Chen, Analytical Biochemistry 417 (2011) 89.[54] W. Yi, W. Liang, P. Li, S. Li, Z. Zhang, M. Yang, A. Chen, B. Zhang, C. Hu, Biotech-

nology Letters 33 (2011) 1539.[55] W. Chunglok, P. Khownarumit, P. Rijiravanich, M. Somasundrum, W. Surare-

ungchai, Analyst 136 (2011) 2969.[56] A. Hayat, L. Barthelmebs, J.-L. Marty, Analytica Chimica Acta 690 (2011) 248.[57] F. Li, L. Mei, Y. Li, K. Zhao, H. Chen, P. Wu, Y. Hu, S. Cao, Biosensors and

Bioelectronics 26 (2011) 4253.[58] S.-P. Chen, X.-D. Yu, J.-J. Xu, H.-Y. Chen, Biosensors and Bioelectronics 26

(2011) 4779.[59] B. Zhang, D. Tang, B. Liu, H. Chen, Y. Cui, G. Chen, Biosensors and Bioelectronics

28 (2011) 174.[60] L.-G. Zamfir, I. Geana, S. Bourigua, L. Rotariu, C. Bala, A. Errachidc, N. Jaffrezic-

Renault, Sensors and Actuators B 159 (2011) 178.[61] P. Salek, L. Korecka, D. Horak, E. Petrovsky, J. Kovarova, R. Metelka, M. Cadkova,

Z. Bilkova, Journal of Materials Chemistry 21 (2011) 14783.[62] O. Laczka, J.-M. Maesa, N. Godino, J. del Campo, M. Fougt-Hansen, J.P. Kutter, D.

Snakenborg, F.-X. Munoz-Pascual, E. Baldrich, Biosensors and Bioelectronics26 (2011) 3633.

[63] M. Hervas, M.A. Lopez, A. Escarpa, Analyst 136 (2011) 2131.[64] A. Ambrosi, M. Guix, A. Merkoci, Electrophoresis 32 (2011) 861.[65] D. Du, J. Wang, L. Wang, D. Lu, J.N. Smith, C. Timchalk, Y. Lin, Analytical Chem-

istry 83 (2011) 3770.[66] Q. Li, L. Zeng, J. Wang, D. Tang, B. Liu, G. Chen, M. Wei, ACS Applied Materials

& Interfaces 3 (2011) 1366.[67] M. de Souza Castilho, T. Laube, H. Yamanaka, S. Alegret, M.I. Pividori, Analytical

Chemistry 83 (2011) 5570.[68] T. Laube, S.V. Kergaravat, S.N. Fabiano, S.R. Hernandez, S. Alegret, M.I. Pividori,

Biosensors and Bioelectronics 27 (2011) 46.[69] Q. Li, D. Tang, J. Tang, B. Su, G. Chen, M. Wei, Biosensors and Bioelectronics 27

(2011) 153.[70] J. Tang, D. Tang, R. Niessner, G. Chen, D. Knopp, Analytical Chemistry 83 (2011)

5407.[71] N.V. Panini, E. Salinas, G.A. Messina, J. Raba, Food Chemistry 25 (2011)

791.[72] Y. Piao, Z. Jin, D. Lee, H.-J. Lee, H.-B. Na, T. Hyeon, M.-K. Oh, J. Kim, H.-S. Kim,

Biosensors and Bioelectronics 26 (2011) 3192.[73] M. Szymanski, R. Porter, V. Gowri, Y. Dep, B.G.D. Wang, Haggett, Physical

Chemistry Chemical Physics 13 (2011) 5383.[74] W. Zhan, A.J. Bard, Analytical Chemistry 79 (2007) 459.[75] T.L. Pittman, B. Thomson, W. Miao, Analytica Chimica Acta 632 (2009) 197.[76] M. Li, Y. Sun, L. Chen, L. Li, G. Zou, X. Zhang, W. Jin, Electroanalysis 22 (2010)

333.[77] N. Gan, J. Hou, F. Hu, Y. Cao, T. Li, Z. Guo, J. Wang, Sensors 11 (2011) 7749.[78] J. Hou, N. Gan, F. Hu, L. Zheng, Y. Cao, T. Li, International Journal of Electro-

chemical Science 6 (2011) 2845.[79] J. Qiu, H. Peng, R. Liang, Electrochemistry Communications 9 (2007) 2734.[80] S. Qu, F. Huang, G. Chen, S. Yu, J. Kong, Electrochemistry Communications 9

(2007) 2812.[81] S. Qu, J. Wang, J. Kong, P. Yang, G. Chen, Talanta 71 (2007) 1096.[82] G.-S. Lai, H.-L. Zhang, D.-Y. Han, Microchimica Acta 165 (2009) 159.[83] M. Pita, T.K. Tam, S. Minko, E. Katz, ACS Applied Materials & Interfaces 1 (2009)

1166.[84] R.S.J. Alkasir, M. Ganesana, Y.-H. Won, L. Stanciu, S. Andreescu, Biosensors and

Bioelectronics 26 (2010) 43.[85] M.I. Kim, Y. Ye, B.Y. Won, S. Shin, J. Lee, H.G. Park, Advanced Functional Mate-

rials 21 (2011) 2868.[86] S. Chawla, C.S. Pundir, Biosensors and Bioelectronics 26 (2011) 3438.[87] X. Chen, J. Zhu, Z. Chen, C. Xu, Y. Wang, C. Yao, Sensors and Actuators B 159

(2011) 220.[88] S. Wu, H. Wang, S. Tao, C. Wang, L. Zhang, Z. Liu, C. Meng, Analytica Chimica

Acta 686 (2011) 81.[89] B. Perez-Lopez, A. Merkoci, Advanced Functional Materials 21 (2011) 255.[90] V.H. Sima, S. Patris, Z. Aydogmus, A. Sarakbi, R. Sandulescu, J.-M. Kauffmann,

Talanta 83 (2011) 980.[91] Z.-G. Xiong, J.-P. Li, L. Tang, Z.-Q. Chen, Chinese Journal of Analytical Chemistry

38 (2010) 800.

[92] A.F. Martin, T.A. Nieman, Biosensors and Bioelectronics 12 (1997) 479.[93] G. Liu, Y. Lin, Journal of the American Chemical Society 129 (2007) 10394.[94] M. Masarik, K. Cahova, R. Kizek, E. Palecek, M. Fojta, Analytical and Bioanalyt-

ical Chemistry 388 (2007) 259.[95] H. Zhang, S.M. Mitrovski, R.G. Nuzzo, Analytical Chemistry 79 (2007) 9014.

a Acta 84 (2012) 62– 73 73

[96] H. Hanaee, H. Ghourchian, A.-A. Ziaee, Analytical Biochemistry 370 (2007)195.

[97] A. Erdem, F. Sayar, H. Karadeniz, G. Guven, M. Ozsoz, E. Piskin, Electroanalysis19 (2007) 798.

[98] S. Hason, H. Pivonkova, V. Vetterl, M. Fojta, Analytical Chemistry 80 (2008)2391.

[99] X. Mao, G. Liu, S. Wang, Y. Lin, A. Zhang, L. Zhang, Y. Ma, ElectrochemistryCommunications 10 (2008) 1847.

[100] P. Du, H. Li, W. Cao, Biosensors and Bioelectronics 24 (2009) 3223.[101] P. Horakova, E. Simkova, Z. Vychodilova, M. Brazdova, M. Fojta, Electroanalysis

21 (2009) 1723.[102] X. Zhang, H. Su, S. Bi, S. Li, S. Zhang, Biosensors and Bioelectronics 24 (2009)

2730.[103] S. Laschi, I. Palchetti, G. Marrazza, M. Mascini, Bioelectrochem 76 (2009) 214.[104] F. Berti, S. Laschi, I. Palchetti, J.S. Rossier, F. Reymond, M. Mascini, G. Marrazza,

Talanta 77 (2009) 971.[105] X. Zhang, L. Li, L. Li, J. Chen, G. Zou, Z. Si, W. Jin, Analytical Chemistry 81 (2009)

1826.[106] N. Gan, X. Yang, D. Xie, Y. Wu, W. Wen, Sensors 10 (2010) 625.[107] K. Nemcova, L. Havran, P. Sebest, M. Brazdova, H. Pivonkova, M. Fojta, Ana-

lytica Chimica Acta 668 (2010) 166.[108] S. Laschi, R. Miranda-Castro, E. Gonzalez-Fernandez, I. Palchetti, F. Reymond,

J.S. Rossier, G. Marrazza, Electrophoresis 31 (2010) 3727.[109] F. Yu, G. Li, B. Qu, W. Cao, Biosensors and Bioelectronics 26 (2010) 1114.[110] E.E. Ferapontova, M.N. Hansen, A.M. Saunders, S. Shipovskov, D.S. Sutherland,

K.V. Gothelf, Chemical Communications 46 (2010) 1836.[111] Y.-H. Bai, J.-Y. Li, J.-J. Xu, H.-Y. Chen, Analyst 135 (2010) 1672.[112] P. Geng, X. Zhang, Y. Teng, Y. Fu, L. Xu, M. Xu, L. Jin, W. Zhang, Biosensors and

Bioelectronics 26 (2011) 3325.[113] Y. Zhang, G.-M. Zeng, L. Tang, Y.-P. Li, L.-J. Chen, Y. Pang, Z. Li, C.-L. Feng, G.-H.

Huang, Analyst 136 (2011) 4204.[114] K. Li, Y. Lai, W. Zhang, L. Jin, Talanta 84 (2011) 607.[115] O.A. Loaiza, E. Jubete, E. Ochoteco, G. Cabanero, H. Grande, J. Rodriguez,

Biosensors and Bioelectronics 26 (2011) 2194.[116] M.-J. Li, Z. Chen, V.W.-W. Yam, Y. Zu, ACS Nano 2 (2008) 905.[117] E. Torres-Chavolla, E.C. Alocilja, Biosensors and Bioelectronics 26 (2011) 4614.[118] Y.-B. Tang, D. Xing, D.-B. Zhu, J.-F. Liu, Analytica Chimica Acta 582 (2007) 275.[119] X. Zhou, D. Xing, D. Zhu, L. Jia, Electrochemistry Communications 10 (2008)

564.[120] D. Zhu, D. Xing, Y. Tang, L. Zhang, Biosensors and Bioelectronics 24 (2009)

3306.[121] X. Zhou, D. Xing, D. Zhu, Li Jia, Analytical Chemistry 81 (2009) 255.[122] J. Wei, B. Wu, Sensors and Actuators B 139 (2009) 429.[123] H. Zhou, D. Xing, D. Zhu, X. Zhou, Talanta 78 (2009) 1253.[124] D. Zhu, J. Liu, Y. Tang, D. Xing, Sensors and Actuators B 149 (2010) 221.[125] R. Duan, X. Zhou, D. Xing, Analytical Chemistry 82 (2010) 3099.[126] Q. Su, D. Xing, X. Zhou, Biosensors and Bioelectronics 25 (2010) 1615.[127] Q. Li, X. Zhou, D. Xing, Biosensors and Bioelectronics 26 (2010) 859.[128] X. Hun, H. Chen, W. Wang, Biosensors and Bioelectronics 26 (2011) 3887.[129] Y. Guo, X. Jia, S. Zhang, Chemical Communications 47 (2011) 725.[130] Y. Long, X. Zhou, D. Xing, Biosensors and Bioelectronics 26 (2011) 2897.[131] J. Zheng, W. Feng, L. Lin, F. Zhang, G. Cheng, P. He, Y. Fang, Biosensors and

Bioelectronics 23 (2007) 341.[132] S. Centi, L.B. Sanmartin, S. Tombelli, I. Palchetti, M. Mascini, Electroanalysis

21 (2009) 1309.[133] A. Erdem, H. Karadeniz, G. Mayer, M. Famulok, A. Caliskan, Electroanalysis 21

(2009) 1278.[134] J. Zheng, G.-F. Cheng, P.-G. He, Y.-Z. Fang, Talanta 80 (2010) 1868.[135] L. Bonel, J.C. Vidal, P. Duato, J.R. Castillo, Biosensors and Bioelectronics 26

(2011) 3254.[136] Y. Wang, X. He, K. Wang, X. Ni, J. Su, Z. Chen, Biosensors and Bioelectronics 26

(2011) 3536.[137] L. Barthelmebs, A. Hayat, A.W. Limiadi, J.-L. Marty, T. Noguer, Sensors and

Actuators B 156 (2011) 932.[138] D. Tang, J. Tang, Q. Li, B. Liu, H. Yang, G. Chen, RSC Advances 1 (2011) 40.[139] D. Tang, J. Tang, Q. Li, B. Su, G. Chen, Analytical Chemistry 83 (2011) 7255.[140] J. Zhao, Y. Zhang, H. Li, Y. Wen, X. Fan, F. Lin, L. Tan, S. Yao, Biosensors and

Bioelectronics 26 (2011) 2297.[141] D. Zhu, X. Zhou, D. Xing, Biosensors and Bioelectronics 26 (2010) 285.[142] C. Ding, Y. Ge, S. Zhang, Chemistry-A European Journal 16 (2010) 10707.[143] N. Gan, J. Hou, F. Hu, Y. Cao, T. Li, L. Zheng, J. Wang, International Journal of

Electrochemical Science 6 (2011) 5146.[144] Y. Du, H. Wei, J.Z. Kang, J.L. Yan, X.B. Yin, X.R. Yang, E.K. Wang, Analytical

Chemistry 77 (2005) 7993.[145] R. Pyati, M.M. Richter, Annual Reports on the Progress of Chemistry Section C

103 (2007) 12.[146] S. Centi, S. Tombelli, M. Minunni, M. Mascini, Analytical Chemistry 79 (2007)

1466.