sandwich-type immunosensors and immunoassays exploiting nanostructure labels: a review

Analytica Chimica Acta 758 (2013) 1 18

Contents lists available at SciVerse ScienceDirect

Analytica Chimica Acta

j ourna l ho me page: www.elsev ier .com/ locate /aca

Review

Sandwnanost

Xiaomei Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology forFood Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China

h i g h l

Sandwich-and imnanostruc

Nanolabelimmunose

Nanolabelimmunose

Nanolabelimmunose

Nanolabelimmunoas

a r t i c l

Article history:Received 11 SeReceived in reAccepted 30 OAvailable onlin

Keywords:ImmunosensoImmunoassaySandwich assaNanoparticle l

Contents

1. Introd2. Immu

2.1.

CorresponPR China. Tel.:

E-mail add

0003-2670/$ http://dx.doi.oi g h t s

type immunosensorsmunoassays exploitingture labels.-based electrochemicalnsing and immunoassay.-based opticalnsors and immunoassays.-based mass-sensitivensing.-based multianalytesays.

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ptember 2012vised form 25 October 2012ctober 2012e 9 November 2012

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y modeabel

a b s t r a c t

Methods based on sandwich-type immunosensors and immunoassays have been developed for detectionof multivalent antigens/analytes with more than one eptiope due to the use of two matched anti-bodies. High-afnity antibodies and appropriate labels are usually employed for the amplication ofdetectable signal. Recent research has looked to develop innovative and powerful novel nanoparticlelabels, controlling and tailoring their properties in a very predictable manner to meet the require-ments of specic applications. This articles reviews recent advances, exploiting nanoparticle labels,in the sandwich-type immunosensors and immunoassays. Routine approaches involve noble metalnanoparticles, carbon nanomaterials, semiconductor nanoparticles, metal oxide nanostructures, andhybrid nanostructures. The enormous signal enhancement associated with the use of nanoparticle labelsand with the formation of nanoparticle-antibody-antigen assemblies provides the basis for sensitivedetection of disease-related proteins or biomolecules. Techniques commonly rely on the use of biofunc-tionalized nanoparticles, inorganic-biological hybrid nanoparticles, and signal tag-doped nanoparticles.Rather than being exhaustive, this review focuses on selected examples to illustrate novel concepts andpromising applications. Approaches described include the biofunctionalized nanoparticles, inorganic-biological hybrid nanoparticles, and signal tage-doped nanoparticles. Further, promising application inelectrochemical, mass-sensitive, optical and multianalyte detection are discussed in detail.

2012 Elsevier B.V. All rights reserved.

uction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2nosensor and immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Immunosensor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

ding author at: Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education), Department of Chemistry, Fuzhou University, Fuzhou 350108, +86 591 2286 6125; fax: +86 591 2286 6135.ress: [email protected] (D. Tang).

see front matter 2012 Elsevier B.V. All rights reserved.rg/10.1016/j.aca.2012.10.060ich-type immunosensors and immunoassays exploitingructure labels: A review

Pei, Bing Zhang, Juan Tang, Bingqian Liu, Wenqiang Lai, Dianping Tang

2 X. Pei et al. / Analytica Chimica Acta 758 (2013) 1 18

2.2. Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43. Nanolabels-based sandwich-type immunosensor and immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.1. Electrochemical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.2. Mass-sensitive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

. . . . . . . . . . .

. . . . . . . . . . . .. . . . . .

3.4. . . . . . .4. Concl . . . . .

Ackno . . . . . .Refer . . . . . .

Bingqian Liu is currently a PhD candidate at theDepartment of Chemistry, Fuzhou University, China.She obtained her Bachelor degree at the Departmentof Chemistry, Shandong University in 2010. Her mainresearch interests focus on pharmaceutical analysisusing nano- and electrochemical immunosensors andimmunoassays.

Wenqiang Lai is currently a PhD candidate at theDepartment of Chemistry, Fuzhou University, China.He obtained her Bachelor degree at the Departmentof Chemistry, Fuzhou University in 2011. His mainresearch interests focus on nano- and electrochemicalimmunosensors and immunoassays.

Dianping Tang is currently a Full Professor at theDepartment of Chemistry, Fuzhou University, China.He obtained his PhD in Analytical Chemistry at South-west University of China (2008). His research focuseson analytical and bioanalytical chemistry, nano-and electrochemical biosensors, clinical immunoas-say, and synthesis and application of multifunctionalnanomaterials. He coordinates projects related toclinical, food and environmental immunoassays.

1. Introdu

Sensitiveteins is essand food ausing certathat speciate a target3.3. Optical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.3.1. Chemiluminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.3.2. Electrochemiluminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.3.3. Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.3.4. Surface plasmon resonance (SPR) . . . . . . . . . . . . . . . . . . . . . . . . . . Multianalyte immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

uding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .wledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Xiaomei Pei obtained her PhD degree at the Depart-ment of Chemistry, Fuzhou University in 2011.Now she is working as a teacher at the Depart-ment of Chemistry, Jiangnan University, China. Hermain research interests focus on supramolecularassemblies, intermolecular interaction analysis andelectrochemical immunosensors.

Bing Zhang is currently a PhD candidate at theDepartment of Chemistry, Fuzhou University, China.She obtained her Bachelor degree at the Departmentof Chemistry, Liaocheng University in 2010. Her mainresearch interests focus on nano- and electrochemicalimmunosensors and immunoassays.

Juan Tang is currently a PhD candidate at the Depart-ment of Chemistry, Fuzhou University, China. Sheobtained her Bachelor degree at the Department ofChemistry, Shangrao Normal University in 2009. Hermain research interests focus on chemical sensors andbiosensors.ction

and specic determination of biomolecules and pro-ential in clinical diagnosis, environmental evaluationnalysis [1,2]. Typically, the assay is performed by

in afnity ligands comprising aptamers and antibodiescally interact with the biomolecules and thus medi--responsive signal transduction cascade [3]. Nowadays,

particular immunosenspecicity immunoasstal microbaand chemilstudied on complex in. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16attention has been paid to using immunoassays orsors due to their advantages of high sensitivity and[4,5]. And more sophisticated analytical devices foray, such as surface plasmon resonance, quartz crys-lance, optical detection methods including uorescenceuminescence, and electrochemical method, have beenthe basis of various signal generation principles fromteractions between antibodies and antigens [6,7]. The

X. Pei et al. / Analytica Chimica Acta 758 (2013) 1 18 3

Fig. 1. Sandwture labels.

assay modetype assay.the antigenpete with tdetectable stion, which[8]. In contrsandwich-tspecicity bThe measuis directly psample, thuthe increasiis one of thimmunoassthe change paratively lusually emp

Typical enzyme labproduct [12of enzyme immunoassrapidly emecesses usedprovides exnew analytiusing nanolor their proas high surfparison witprovide unwith bulk mgood biocooptical prop

Various carbon nannanostructuin the sand[15]. Antibo

their bioactivity and interact with their counterparts, and basedon the detection of those nanoparticles, the amount or concentra-tion of analytes can be determined [16,17]. The enormous signal

ement associated with the use of nanomaterial amplifyingand lies munwith

nanon. Th

can hat oplic

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uno

mun

unos thaucersnd ailizarme

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detet) [2ich-type immunosensings and immunoassays exploiting nanostruc-

s mainly contain sandwich-type assay and competitive- In a competitive format, unlabeled analyte (usually) in the test sample is measured by its ability to com-he labeled antigen in the immunoassay. Typically, theignal decreases with the increase of analyte concentra-

requests a high background signal toward zero analyteast, noncompetitive immunoassay formats (usually theype formats) give the highest level of sensitivity andecause of the use of a couple of match antibodies [9].

rement of the labeled analyte (usually the antibody)roportional to the amount of antigen present in thes resulting that the detectable signal increases withng target analyte [10]. Therefore, sandwich-type assaye most popular schemes in the immunosensings andays. Although the antigenantibody reaction can causeof detectable signal to some extent, the change is com-ittle. High-afnity antibodies and appropriate labels areloyed for the amplication of detectable signal [11].

enhanclabels assemband imreacts on thereactiobodieswhile tfor am

Dueit is this focusepromissandwnanolasays intheir s

2. Imm

2.1. Im

Immdevicetransdment aimmobtransfomeritsand afon thedividedtrocheused, ttion isinducesitivelyinteresmethods involve the use of an indicator system (e.g.el) that results in the amplication of the measured]. Since there is, for sterical reasons, usually a 1:1 ratioand signal antibody used in the traditional enzymeays, the detectable signal is always limited [13]. Therging research eld of nanoparticle labels, and the pro-

to generate, manipulate and deploy nanomaterials,citingly new possibilities for advanced development ofcal tools and instrumentation. One major advantage inparticle labels lies in the possibility to control and tai-perties to meet the needs of specic applications, suchace-to-volume ratio and unique conductivity, in com-h bulk materials [14]. For example, nanomaterials canique chemical and physical properties (in comparisonaterials) enabling new and advanced functions such as

mpatibility, high surface-to-volume ratio, and uniqueerties.

nanoparticle labels including noble metal nanoparticles,omaterials, semiconductor nanoparticles, metal oxideres, and hybrid nanostructures, have been developedwich-type immunosensors and immunoassays (Fig. 1)dies (antigens) labeled with nanoparticles can retain

the terms dare tracer-binding is dis bound toto an immoplace after secondary immobilize

For the problem ofpied bindinsandwich-tally immobimmunocomantibodies bodies or nmainly deradvances infor improviimmunoassmodern meIn this regwith the formation of nanoparticleantibodyantigenprovides the basis for ultrasensitive immunosensingsoassays [18]. When one antibody on the nanoparticle

the corresponding antigen, other biomolecules labeledparticle will be carried over, and thus participate in theerefore, the high immobilized amount with the anti-increase the possibility of antigenantibody reaction,f the enzymes can enhance the measurable sensitivityation of detectable signal.the overwhelming amount of literature available,choice to underline the most recent trends inFig. 2). Rather than being exhaustive, this review

selected examples to illustrate novel concepts andapplications using nanoparticle-based labels in theype immunosensors and immunoassays. We dividebased sandwich-type immunosensors and immunoas-ectrochemical, mass-sensitive and optical, according to-transduction methods.

sensor and immunoassay

osensor

sensors are afnity ligand-based biosensing solid-statet couple immunochemical reactions to appropriate. Generally, an immunosensor consists of a sensing ele-

transducer. The sensing element is formed by means oftion of antigens or antibodies, and the binding event isd into a measurable signal by the transducer [19]. Themunosensors are obviously related to the selectivity

of the antibodyanalyte binding reaction. Dependingsducer technology employed, immunosensors can be

three principal classes: optical, piezoelectric and elec-l. Furthermore, on the basis of the immunoassay formatan be either direct (where the immunochemical reac-ctly determined by measuring the physical changesthe formation of the complex) or indirect (where a sen-ctable label is combined with the antibody or antigen of

0]. The distinction has an entirely different meaning asirect and indirect in the immunoassay eld, which bothrelated. They are distinguished by whether antibodyirectly detected, e.g. after an enzyme-labeled antibody

an immobilized coating conjugate or an enzyme tracerbilized antibody, or whether the detection only takesa secondary binding reaction, e.g. if an enzyme-labeledantibody is used to label a rs antibody bound to and coating conjugate [2123].indirect assay modes, it signicantly facilitates the

signal generation. The tracer either labels the occu-g sites of the antibodies or the free ones. As for theype immunosensors, the primary antibodies are usu-ilized on a solid-state support, and the sandwichedplex is formed between the immobilized primary

and signal antibodies (usually enzyme-labeled anti-anoparticle-labeled antibodies). The detectable signalives from the labeled signal tags. In spite of many

this eld, there is still a paucity of novel approachesng the simplicity, selectivity, and sensitivity of clinicalays, in order to respond to the demands and needs ofdical diagnostics and biomedical research applications.ard, the protein-mediated assembly of nanoparticles


mun

is a potenimmunosenfeatures (siunique phy

2.2. Immun

Immunoity and spebeen considiagnostic based on tsor is specimmunoreasists of hetheterogenebilized on aimmunoasswith homosays are eaa step of sneed to imface. In conin the immbeads, and tof multiplebining withcan be usedor blood, wpretreatmemance. Micof portabilittechnologiethe unproce

3. Nanolabimmunoas

3.1. Electro

Electrocin a solutioor a semicowhich involtrolyte or spmethods poand high cogies, they h

oassthe ely lal imeducriatechemectioent seaner inetweuing aledReceeviseal sie lab

pues (iactivlene 438horizn [39atly ectrindum dy the]. Baomppet

se wl-offary. IFig. 2. Number of published articles relative to the immunosensor and im

tial tool for the fabrication of new sandwich-typesors. This approach combines tunable nanoparticleze, surface functionality, and core properties) with thesical and chemical properties of proteins and peptides.

oassay

assays are analytical techniques based on the avid-cicity of the antigenantibody reaction, and it hasdered as one of the most widely used biomedicalmethods. The term immunoassay is used for testshe immunoreactions, while the term immunosen-ically employed to describe whole instruments, i.e.ction-based biosensors. Usually, the technique con-erogeneous and homogeneous immunoassays. In theous immunoassays, the antibody or antigen is immo-

solid substrate (e.g. microplate), while homogeneousays take place in the solution phase [24]. Comparedgenous immunoassays, the heterogeneous immunoas-sily designed and constructed. However, they requireeparating antigen or antibody from the samples andmobilize the antibody or antigen on the solid sur-trast, the homogeneous immunoassays usually involveobilization of the biomolecules on the nano-/micro-ake place in the solution, thus allowing the integration

liquid handling processes [25,26]. Especially com- microuidic device, the homogeneous immunoassay

for the detection of complex samples, such as urineithout the large sample consumption and sample

nt, resulting in a relatively inexpensive and easy perfor-rouidic lab-on-a-chip technology has the advantagesy, integration, and automation. The combination of twos leads to a pathway of point-of-care diagnostics usingssed serum samples.

immuncause relativchemicnoise rappropelectroas detsuremanalyta furthratio bcontinnanosctivity. been dchemicenzymon theenzymelectromethyions [3a new catiobe greand elsemicoquantuamplif[4144on a cthe comdecreaa signanecessels-based sandwich-type immunosensor andsay

chemical

hemistry studies chemical reactions which take placen at the interface of an electron conductor (a metalnductor) and an ionic conductor (the electrolyte), andve electron transfer between the electrode and the elec-ecies in solution. Just as the electrochemical detectionssess high sensitivity, low cost, low power requirement,mpatibility with advanced micromachining technolo-ave extensively applied in the immunosensings and

signal-enhaconcentratiwith more preferable, cated fromor nanoparcoupling wsignal-geneimmunosenometric, po

Amperomost populmethod is uoassay during the period from 2002 to 2011.

ays [27]. Although the antigenantibody reaction canchange in the electrochemical signal, the change isittle. For the successful development of an electro-munosensor or immunoassay, signal amplication andtion are very crucial [28]. High-afnity antibodies and

labels are usually employed for the amplication ofical signal. Enzyme-labeled antibodies are often used

n antibodies that result in amplication of the mea-ignal [29]. However, the association constant of smalltibody complexes may be as high as 10101012 M1, andcrease is almost impossible owing to the limited 1:1en the detection antibody and the enzyme label. Hence,worldwide effort has been made in developing novel

labels capable of providing a highly detectable sensi-ntly, various immunosensors and immunoassays haved and developed for the amplication of the electro-gnal. Routine approaches consist of ligand-conjugatedels and metal-containing nanolabels [3033]. Based

blished papers, the labels mainly contain bioactive.e. horseradish peroxidase and alkaline phosphatase),e materials (e.g. thionine, ferrocene derivatives, andblue), nanostructures, quantum dots (QDs), and metal]. In contrast, the emergence of nanotechnology openson for the use of nanomaterial labels for signal ampli-,40]. The power and scope of such nanomaterials canenhanced by coupling them with immunoreactionscal processes (i.e. nanobioelectronics). Metal and/orctor nanostructures, e.g. gold, carbon tubes, silver, andots, have been directly used as electroactive labels to

signal in the electrochemical detection of proteinssed on the present reports, the assay is carried out basedetitive-type/sandwich-type immunoassay format. Foritive-type immunoassay format, the detectable signalsith the increment of analyte concentrations, and exhibit

tendency. In this case, a strong background signal isn contrast, the sandwich-type immunoassays display a

ncement (i.e. signal-on) mode with the target analyteon increased. Therefore, for the multivalent antigensthan one epitope, the sandwich-type immunoassay isespecially for the low-concentration analyte. As indi-

Fig. 3, however, the number of using pure enzymesticles as labels was less than that of enzyme labelsith nanolabels in the recent 10 years. Based on theration principles, the sandwich-type electrochemicalsors and immunoassays are mainly classied as amper-tentiometric, impedimetric and capacitometric.metric immunosensors and immunoassays are one ofar approaches during the electrochemical process. Thesually designed to measure current generated by the


Fig. 3. The pthe sandwich-period from 20

electrochemric methodselectrode isSince mosttically act ahowever, acal reactioncommerciasis of the entoward appHowever, tusefulness tion has beelabels or msignal reporDNA-basedpolymeraseture antibodwith a highimmunoassthe comple

Nanoteccovers diveengineeringingly new pmethods anapplicationsynthesizedand applicaogy diagnocountries [6terial labels[15]. Thesenanosilica, so on. Goldhave manyanalytical agroup utilizcytochemiswith the band DNA. Gnanoparticlitoring of pgold nanop

surface free energy could enhance the sensitivity of the ampero-metric immunoassays. Recently, this method was further extendedfor amperometric immunosensing of alpha-fetoprotein (AFP) onthionine/nanogold multilayer lm-functionalized immunosensor

iofu-conit (Lat o. In interleculng inuble-les o

e ratiion oent r

dispere uomet

golbelsce [6e as

higharticlcal nn ththat

cou spe

theohanic acn (No impto pcal coower]. Thre anctrooweric edentlyneti

opedologercentage of published articles relative to the different labels intype electrochemical immunosensors and immunoassays during the02 to 2011.

ical reaction [45,46]. Most of the present amperomet- are a subclass of voltammetric analyses in which the

held at constant potentials for various lengths of time. analytes (e.g. antigens or antibodies) cannot intris-s the redox partners in an electrochemical reaction,n electroactive label is needed for the electrochemi-

of the analyte at the sensing electrode [4749]. Mostlly available immunoassays were based on the cataly-zyme-labeled secondary antibodies (e.g. HRP and ALP)ropriate substrates to form electroactive products [50].he sensitivity is still limited, which restricts the widein the early diagnosis of disease. Recently, great atten-n focused on signal amplication using bionanoparticleultienzyme labels, and employing DNA as an ampliedter in the sandwich-type immunoassays [5156]. In the

immunoassays, the signal was usually amplied using chain reaction (PCR) after target recognition of cap-y, whereas it was pre-amplied by using nanoparticles

ratio of DNA to capture antibody in the bio-barcodeay [5759]. Thus, their application is restricted due tox detection procedures or conjugation processes.hnology is multidisciplinary and interdisciplinary andrse elds including chemistry, physics, material science,, biology, and even medicine [60]. It provides excit-ossibilities for advanced development of new analyticald instruments for bioanalytical and biotechnological

using bof HRPtion limthan thbodiesas an biomoresultithe domolecuvolumreduct

Recshapesthey wampershapedtrace lainterfacles, thexhibitnanopspheribetweefound trationRamandue to[67]. Mcatalytreactioare twbonds spherinanoity [69structuthe elenanometall

Recas magions-dotechns. Currently, a vast library of nanostructures has been and documented, with a wide variety of propertiestion. Hauch and co-authors reviewed nanotechnol-stics for infectious diseases prevalent in developing1]. Liu and Lin summarized recent advances in nanoma-

in electrochemical immunosensors and immunoassays nanolabels mainly consisted of nanogold, nanosilver,semiconductor nanoparticles, carbon nanotubes and

nanoparticles (AuNPs), as a class of nanomaterial, unique properties and have been widely used fornd biomedical purposes. In the early 1970s, Hayattsed gold colloids as electron-dense probes in immuno-try [62]. With surface modication, AuNPs can bindiomolecules including peptides, enzymes, antibodiesonzalez-Garcia [63] and Dequaire [64] employed goldes as an electrochemical label for voltammetric mon-rotein interaction. Experimental results revealed thatarticles with high volume-to-surface ratio and strong

enzyme imfrom the lies. Signicdetection aenzyme-dotrocatalyticsurface modthere are mthesized bientered andone antibogen. Tang aimmunoseners using t[7], and HRP[10]. This hsensitive bior signal anctional double-codied gold nanoparticles as the labeljugated anti-AFP secondary antibodies [65]. The detec-OD) of using the bionanolabels could be 10-fold lowerbtained using conventional HRP-labeled anti-AFP anti-this case, the doped gold nanoparticles might severvening spacer matrix to extend the immobilizedes away from the substrate matrix in the mobile phase,

binding sites more accessible to antigens. Meanwhile,codied gold nanolabels contain many HRPanti-AFPn each nanoparticle surface due to the high surface-to-o of nanogold particles, and thus enhance the catalyticf H2O2.esearch indicated that gold nanoparticles with variouslayed different electrochemical characteristics whensed as the label probes. Tangs group designed a newric immunosensor for detection of AFP using irregular-d nanoparticle-labeled HRPanti-AFP conjugates as

on carbon nanoparticle-functionalized immunosensing6]. Compared with same-size spherical gold nanoparti-say of using irregular-shaped gold nanoparticles coulder current responses. Maybe, the irregular-shaped goldes could display stronger zigzag effect than that ofanoparticles, and improve the electron communicatione nanoparticles. In addition, Choi and co-workers alsothe effect of chemotherapeutic agents at low concen-ld be successfully detected based on surface-enhancedctroscopy (SERS) technique and cyclic voltammetry

increased sensitivity provided by gold nanoowerstys group reported that Pt nanoowers have superiortivity for the Suzuki-Miyaura and the Heck couplingote: Suzuki-Miyaura and the Heck coupling reactionortant noble-metal-catalyzed processes for forming CC

roduce medicines, agrochemicals and fragrances.) overunterparts [68]. Jena and Raj demonstrated that golds exhibited pronounced SERS and electrocatalytic activ-e ower-like nanoparticles possessed unique surfaced high surface-to-volume ratio, which might improve

chemical behavior of immunosensor [70]. Meanwhile,s in the zig-zag orientation could show spin-polarizedge currents., various types of the doped nanostructures, suchc particles-, semiconductor quantum dots and metal

nanostructures, have preliminarily applied in bionan-y and biomedicine. For the conventional sandwich-typemunoassays, the detection signal usually derives

abeled enzyme conjugated with secondary antibod-antly, if more enzyme molecules are labeled to thentibodies, the sensitivity might be enhanced. Bioactiveped nanoparticles possess several merits: high bioelec-

intensity, good biocompatibility, and good potential forication with various biomolecules. Using this method,any enzyme molecules inside and outside of the syn-onanolabels. The carried enzyme molecules will be

participated in the catalytic reaction, suspecting, whendy among them reacts with the corresponding anti-nd co-workers developed two types of amperometricsor for ultrasensitive determination of cancer biomark-hionine-doped magnetic gold nanoparticles as labels-encapsulated nanogold hollow microspheres as labels

igh efciency makes them especially suitable for ultra-oanalysis, and negates the need for additional reagentsmplication steps. Just as the advantages of hybrid


nanostructures, various hybrid nanomaterials have been used asthe labels for the development of sandwich-type amperometricimmunosensors and immunoassays, e.g. nanogold-functionalizedmagnetic beads [13], poly(o-phenylenediamine)-carried nanogoldparticles [5and so on. usually immniques. In tNanoparticsensitive antions withopolymerase

Usually,of particleclonal antibnanoparticlantibodies elements. Mseparation applied in tsays, proteidelivery. Thmagnetic napplying a them fromMirkin and for the detattomolar cmicroparticthe DNA-conanoparticlat concentrchip using assay allownal amplicowing to thgroup devisimmunoassmagnetic ime.g. enzyme[74] and na

Howevethe labeledlabeled ontissue, someof enzymesnanostructureactions osuch as Fe3ites, Pd nanfor catalyticAmong thesfound to plalow-tempeor nitrogenAuNPs canaminophenthe produc(QI) with tagain to APreactants rnal. Tang aelectrochemof AFP usinanolabels/improved b

iridium oxide nanospheres [82] with catalytic recycling of self-produced reactants. Moreover, the enzyme-free electrochemicalimmunosensors and immunoassays could also be fabricated byusing quantum dots [83] or metal ions-doped nanostructures [84]

labelridiz

n rols, theuctiomplion w

haioduc

to polyl an

of lcatioal s

(Figion o

cape sewith

initirpin

gnetiode./G-quand bosen

s to aeledentiontia

ion dve aelectutioncal c

antibains]. Iments aractstruep [9cal cothateth

munrepon of -worsing[98].uantich iere s wa neipet

ion li sand

IgGpli1], nanogoldpolyanilinenanogold microspheres [71]Among these methods, the primary antibodies wereobilized on the modied electrodes using various tech-

his case, it is difcult to realize the continuous testing.les-based assays hold great promise in realizing highlyd selective detection at attomolar protein concentra-ut requiring complex amplication methods including

chain reaction (PCR). the nanoparticles-based assay consisted of two kindss: (i) magnetic microparticles coated with mono-odies as immunosensing probe and (ii) multifunctionales (i.e. nanogold particles) decorated with polyclonalor unique barcode DNA sequences as recognitionagnetic beads with good biocompatibility and rapidfrom the substrate solution have been extensivelyhe elds of DNA hybridization detection, immunoas-n and enzyme immobilization, cell separation, and druge functionalized probes could pull antibodies bound toanoparticles from one laminar ow path to another bylocal magnetic eld gradient and selectively remove

owing biological uids without any washing step.his colleagues reported a nanoparticle-based approachection of free prostate-specic antigen (PSA) at lowoncentration by adding an antibody-labeled magneticle, DNA barcodes, and conjugating a second antibody tonjugated gold nanoparticle [72]. Lius group reported aes-based assay for the highly sensitive PSA detectionation as low as 500 attomolar on a single disposablelight scattering method [73]. The nanoparticles-baseds for the detection of low concentration levels with sig-ation, and reduces sample pretreatment requiremente presence of magnetic particles. Recently, the Tanged several nanoparticle-based sandwich amperometricays for detection of biomarkers on the functionalizedmunosensing probes based on the different nanolabels,-coated nanometer-sized enzyme-doped silica beadsnogold/graphene nanosheets [26].r, recent experiments found that the bioactivity of

enzymes is usually weakened when enzymes areo the signal antibody or nanostructures. To tackle this

newly amplied strategies without the participation were devised. Nanostructures, especially redox-activeres, are usually used as catalysts for electrochemicalr organic/inorganic synthesis. Various nanocatalysts,O4/MnO2 hybrid nanocrystals, ZnOSiO2 nanocompos-oparticles and gold nanoparticles, have been reported

organic synthesis and electrocatalytic reaction [7578].e nanocatalysts, gold nanoparticles (AuNPs) have beeny an important role in the catalytic processes including

rature CO oxidation, reductive catalysis of chlorinatedated hydrocarbons, and organic synthesis. Signicantly,

catalyze the reduction of p-nitrophenol (NP) to p-ol (AP) in the presence of NaBH4 [79]. Meanwhile,ed AP molecules can be oxidized to p-quinone iminehe help of electron mediators, which can be reduced

via the NaBH4. The catalytic recycling of self-producedesulted in the amplication of electrochemical sig-nd his colleagues reported an enzyme-free sandwichical immunoassay for ultrahighly sensitive detection

ng carbon nanotube-enriched gold nanoparticles asnanocatalysts [80]. Later, this methodology was furthery coupling gold nanoowers [81] and multifunctional

as the Hyb

ductioprocesconstrinto coformatof DNAis intreventsing coa novenationamplichemicprobesformatbilizedand thgated of DNAH2* haof mation mheminalysts immunwork ithe lab

Potor potedetecttion hathe isothe solelectrithat ofcally ag[9395suremcan chnon-detion stelectriaction three mical ima few catioand coteins ulimits CdSe qsandwQDs wminutepH at micropdetectoped ahumannal ams.ation chain reaction (HCR) can also play the trans-e via an amplication approach [8587]. During this

single-stranded DNA (ssDNA) molecule is a versatilen material that can be programmed to self-assembleex structures driven by the free energy of base pairithout enzyme [88,89]. Typically, two stable species

rpins coexist in the solution until an initiator stranded. The initiator triggers a cascade of hybridizationyield nicked double helices analogous to alternat-mers. Zhang et al. reported the proof-of-concept ofd powerful immuno-HCR assay strategy for determi-arger target analytes, e.g. proteins, by coupling then capability of the HCR with the sensitive electro-ignal of ferrocene molecules conjugated to hairpin. 4) [90]. The assay protocol mainly involves thef the sandwiched immunocomplex between the immo-ture antibodies on the magnetic beads (Ab1-MBs)condary antibodies on the gold nanoparticles conju-

initiator strands (Ab2-S1-AuNPs), the HCR reactioniator strands on the Ab2-S1-AuNPs between H1* and

DNA molecules, and electrochemical measurementc immuno-HCR complexes with a sequential injec-

Based on the HCR principle, the same group utilizedadruplex-based DNAzyme concatamers as electrocat-iolabels to construct a sandwich-type electrochemicalsor for sensitive detection of IgG1 [91]. Highlight of thisdequately utilize the signal dual-amplication based on

ferrocene and the formed DNAzyme.metric immunosensors are based on the surface chargel change upon immunoreaction on the interface of theevice. Either antibodies or antigens in aqueous solu-

net electrical charge polarity, which is correlated toric points of the species and the ionic composition of

[92]. If antibody complex combines with antigen, theharge of the resulting complex will be different fromody alone. This change can be measured potentiometri-t the reference electrode immersed in the same solutionpedimetric immunosensors, based on impedance mea-of the electrical equivalent circuit of the oscillator,erize the electrical properties of immunoassay systemsctively without the need for reagents and a separa-6]. Capacitive immunosensors are based on alteringnductivity at a constant voltage, caused by immunore-

specically generates or consumes ions [97]. Theseods are usually adopted in the label-free electrochem-osensors and immunoassays. However, there are stillrts focusing on the nanolabels for the signal ampli-potentiometric or impedance immunosensor. Thurerkers developed a potentiometric immunoassay of pro-

CdSe quantum dot (QD) labels, and


meth(from Ref. [90]

high resistacomplex, thconcentrati

Conductsupport wiin a planar [100]. The of the elecmany biochimmunosento increasienzyme-labconjugatedcomplex coalytic efciesupporting tion can chasignal due ttransductioLiu et al. deatitis B surfon a microcparticles aswere prepaconjugatedthe immunbetween thtivity variatreaction of tH2O2, 0.08 simple and of AFP usinsandwich-tAFP conjugaof AFP in th

crea

ass-s

mas micre thFig. 4. The immuno-HCR assay with permission.)

nce [99]. On the formation of the sandwiched immuno-e resistance decreased with the increment of analyteon.ometric immunosensors, which consist of a planar glassth interdigitated gold electrode pairs on one surfaceconguration, have been introduced by Watson et al.principle of the detection is based on the changestrical resistance between two parallel electrodes by

was deAFP.

3.2. M

Thecrystalmeasuemical reactions in solution. Conductometric enzymesors can detect products of enzymatic reactions dueng conductivity of the enzyme membrane. Wheneled antibodies were immobilized on the electrode and

with antigens in sample solution, the antigenantibodyating on the surface of the electrode inhibits the biocat-ncy of the immobilized enzyme, the conductivity of theelectrolyte was changed. The antigenantibody reac-nge the enzyme activity and hence the immunosensoro the hindrance of access of a substrate or the electronn between the electrode and the enzyme active site.veloped a new conductometric immunoassay for hep-ace antigen (HBsAg) based bioelectrocatalytic reactionomb-type electrode by using double-codied nanogold

labels [101]. The double-codied nanogold particlesred by using nanogold-labeled anti-HBs antibodies

with horseradish peroxidase (HRP). The formation ofocomplex changed the direct electrical communicatione carried HRP and the electrode, and thus local conduc-ions could be assayed based on the bioelectrocatalytiche carried HRP in 0.01 M PBS (pH 7.0) containing 60 MM KI and 0.1 M NaCl. Recently, Tang et al. designed asensitive conductometric immunosensor for detectiong carbon nanoparticles (CNPs) as labels [102]. With aype immunoassay format, the CNP-labeled HRPanti-tes on the transducer were increased with the increasee sample, and the conductivity of the immunosensor

technique, by Sauerbrlinearly proing of QCMfor the casthe resultinshown to b115 A. Whefx and mQCM immuity can be and by decface [1041stabilizatiobody immothree maincrystal precentrapmenglutaraldehimportant ionly be ach

In the Qusually bastages, the uis still encuGreat effortivity of thod.

sed in the H2O2KI system with a LOD of 0.05 ng mL1

ensitive

s-based immunosensors usually involve in the quartzrobalance (QCM) technique. The QCM immunosensorse resonant frequency (f) using the standard oscillator

and the frequency change (fx) is usually explainedey equation, which states that the decrease in f isportional to the increase in surface mass (m) load-

[103]. This Sauerbrey equation, however, holds onlye of rigid coated material. The qualitative aspects ofg data and the magnitude of the observed effect aree independent of lm thickness for values as low asn an overlayer is thick, the relationship between the

is no longer linear and corrections are necessary. Innosensing and immunoassays, the detection sensitiv-improved by increasing the coverage of biomoleculesreasing the steric-hindrance effect on the sensor sur-06]. Thus, antibody immobilization method and its

n are very important. These methods of efcient anti-bilization on QCM immunosensor are classiable into

categories: (i) immobilization of the antibody on theoated with a suitable material; (ii) immobilization viat in polymer membranes; and (iii) immobilization viayde cross-linking [107]. These features are particularlyn the QCM immunosensors because high sensitivity canieved using active, thin and rigid layers.CM immunosensings and immunoassays, the assay ised on a label-free assay format. Despite some advan-se of QCM for determination of trace biological targetmbered by its relatively low intrinsic sensitivity [108].ts have been made to further improve the sensi-e QCM-based immunosensors. At present, there are


two strategies for enhancement of the sensitivity in the mass-sensitive immunosensors. The rst method is the immobilizationof biomolecules mentioned above. Usually, the technique mainlyconsists of adsorption, covalent conjugation, self-assembly, andencapsulatication techmass on thadsorbed athe use of to deposit mass muchsandwich-ttoxin emploliposomes amethod is liTo tackle tamplicatioing by usinthe amplicommerciaand low cotivity was QCM immuby using golon the primThe dendritand enhancIn the preseondary antiresulting inoped for de

In theseon the surfnanoparticlnew possiband instrumticles are atcan be sepamagnetic tion of Eschbased on bgrowth of O157-immusystem andmass enhanof QCM immimmunosen23 CFU mL

analysis tima QCM imm

In additacterizationimmunoassbodies can Liu et al. repnanogold-plabels of sesignal [114complex wathe results trochemicatechnique tperoxidase during the Selective caface increas

in resonance frequency allows measurement of the bindingevent.

3.3. Optical

ical dya

theagesode

beeumin-plaremions ationges uresationed a

to t

Chemmiluroutihes

ativeit an

emild to

intemits enzy. In ydroduce

enzzymee emlar

for Amost w

s rapangent bpose

CL efetop

odiion lit uschiesi grNP)

dieYangg dotopranceampve ang mLloweosorsizedon. Another important strategy is the mass ampli-nique, which comprise the deposition of an ampliede probe surface via a chemical route activated by thenalytical targets. Typical approaches mainly containa catalytic label that induces insoluble precipitateson the probe or the use of a massy label that have

larger than the analyte. Alfonta et al. constructed aype EQCM immunosensor for detection of the choleraying horseradish peroxidase and GM1-functionalizeds the catalytic recognition labels [109]. However, themited due to involving in the bioactivity of the enzymes.his issue, Chu and her colleague devised a dendriticn procedure for sandwich-type QCM immunosens-g nanogold particles-labeled secondary antibodies ascation probes [110]. The assay does not require anyl label reagents, and can be implemented with easyst. Just as the use of nanoparticle labels, the sensi-largely improved. Recently, Xia et al. reported a newnoassay protocol based a double amplication moded nanoparticles-labeled anti-microcystin-LR antibodiesary antibodies-functionalized dendritic surface [111].ic surface increased the surface coverage of the probe,ed the immobilization amount of primary antibodies.nce of target analyte, gold nanoparticles-labeled sec-bodies could be conjugated onto the probe surface, thus

the large frequency shift. The mode was further devel-tection of inuenza by Miller group [112].

methods, the primary antibodies are immobilizedace of gold substrate. The emerging research eld ofe-enabled bio-barcode technology provides excitinglyility for advanced development of new analytical toolsentation for bioanalytical applications. Magnetic par-tractive because they have good biocompatibility andrated very readily from reaction mixtures in an externaleld. Shen et al. described a new method for detec-erichia coli O157:H7 by using a QCM immunosensoreacon immunomagnetic nanoparticles and catalyticcolloidal gold (Fig. 5) [113]. The designed E. colinosensing probes play crucial roles in the detection

have three functions: separation, conjugation, andcement (magnetic nanoparticles). The frequency shiftunosensor is amplied for three times, and the QCM

sor has a high sensitivity, with a detection limit of1 in PBS and 53 CFU mL1 in milk. Moreover, the totale is approximately 4 h. This method of detection usingunosensor can be easily developed and used.ion, the QCM technique is often used as a char-

method in the nanoparticle-labeled sandwich-typeays. The reaction between the antigens and the anti-obviously cause the frequency change of QCM probe.orted a sandwich-type immunoassay method by usingatterned mesoporous CoFe2O4 nanocomposites as thecondary antibodies for the amplication of detectable]. The fabrication process of the sandwiched immuno-s characterized by using the QCM method. Meanwhile,were in accordance with those obtained by the elec-l method. Most recently, Akter et al. also used the QCMo demonstrate carbon nanotube-attached horseradishfor the precipitation process of 4-chloro-1-naphtolformation of the sandwich-type immunoassays [115].pture of an analyte by a functionalized crystal sur-es the effective surface mass, and the ensuing decrease

Optantiboone ofadvantation mIt haschemilsurfacelabels conditinactivchallenprocedconjugexplorapplied

3.3.1. Che

lar in researcthe reltion limthe chmethoto thetion eusing streamwith hto prothe CLthe enogy, thmolecubilities[121]. the mosuch awide rexcelleers proa newalpha-ticles mdetectwithouically aAmbro(DC-Aucle mo[123]. by usinof -fenal enhsignal sensitito 0.3 nmuch immunsyntheimmunosensings and immunoassays combinentigen interaction with optical measurements are

most popular protocols for bioanalysis due to the of applying visible radiation, nondestructive oper-

and the rapid signal generation and reading [116].n developed using different techniques includingescent, electrochemiluminescence, uorescence and

smon resonance (SPR). Creating such nanoparticleains a challenge. Notably, the harsh environmentalinto which these nanoparticles are placed often causes

of the sensitive biological targets. To deal with thesevarious surface modications and immobilization

such as physisorption, afnity interaction, covalent, and entrapment in sol-gel matrices, have been

nd developed (Fig. 6). Further, these have now beenhe conjugation of secondary antibodies.

iluminescenceminescence (CL) methods have become very popu-ne clinical analysis as well in clinical and biomedicalascribed to the advantages as no radioactive wastes,ly simple instrumentation required, the very low detec-d wide dynamic [117119]. CL immunoassay, combing

uminescent systems and the immunoreactions, is a determine the concentrations of samples accordingnsity of the luminescence that the chemical reac-[120]. Nowadays, sandwich-type CL immunosensorsme as label for signal amplication is still the main-the system, the CL reagents in the base solution reactgen peroxide released from the enzymatic reactions

a CL light signal. Therefore, the high sensitivity ofyme immunosensor is determined by the amount of

labeled on the secondary antibody. Bionanotechnol-erging research eld of manipulating matter at the

or atomic level, has provided excitingly new possi-advanced development of CL enzyme immunosensorng these nanomaterials, gold nanoparticles are one ofidely used labels because of their several advantages,id and simple chemical synthesis, easy preparation in a

of sizes, good capability. Thus, they can be used as anio-labeling for CL immunoassay. Zhang and his work-d a novel and sensitive CL immunoassay by employingnhancer, bromophenol blue, for the determination ofrotein based on magnetic beads and gold nanopar-ed with HRP-labeled anti-AFP antibodies [122]. Themit is 1 order of magnitude lower than that obtaineding gold nanoparticles and much lower than that typ-ved by enzyme-linked immunosorbent assay (ELISA).oup reported a novel double-codied gold nanolabel

for detection of human IgG based on gold nanoparti-d with anti-human IgG peroxidase-conjugated antibodys group proposed a similar enhanced CL immunoassayuble-codied gold nanoparticle as labels for detectionotein (AFP) (Fig. 7) [124]. However, a new potential sig-r, 4-(4-iodo)phenylphenol, was introduced for further

lication. The proposed immunoassay presented highd provided a linear response range of AFP from 0.0081 with an extremely low detection limit of 5 pg mL1,r than those achieved by the classical enzyme-linkedbent assay. Recently, irregular gold nanoparticles were

and applied in the CL immunoassay of IgG by Wang


Fig. ic nan(from Ref. [113

and his woluminol CL that of sphonly load anal amplienzyme mimgold metal netic bead-[127]. A larafter oxidadeterminedimmunoassysis has big extremely sor HBrBr2suitable fora CL metalsilver depo

direc2S2Ooma-to-fci

operaon mbes lH2studmplar ra

ion lihan 5. Schematic illustration of QCM immunosensor based on beacon immunomagent] with permission.)

rkers [125]. It is found that the catalytic efciency onof irregular gold nanoparticles is 100-fold greater thanerical gold nanoparticles. Gold nanoparticles can not

large number of enzymes or proteins to achieve sig-cation, but also can directly catalyze CL reactions as

ics [126]. Fan and his works reported a new oxidativedissolution-based CL immunoassay by using a mag-

based CL metal immunoassay with a colloidal gold labelge number of Au3+ from each gold label are releasedtive gold metal dissolution and then quantitatively

by a simple and sensitive Au3+-catalyzed luminol CLay. However, colloidal gold used as the label for CL anal-drawbacks that the dissolution of colloidal gold requiresevere conditions (e.g. highly concentrated HNO3HCl

was inAg+K

Nansurfaceas an ehis cobased nanotuluminoIn the NTs teA lineadetectlower t). Further, Lis group found silver particles were more CL analysis than gold particles. Thus, they proposed

immunoassay for detection of human IgG based onsition on colloidal gold labels [128]. The human IgG

ica nanopaunique proarea, high pmal and m

Fig. 6. Antibody-functionalized nanoparticles with differentoparticles and catalytic growth of gold nanoparticle labels.

tly determined by a sensitive combined CL reaction of8Mn2+H3PO4luminol.terials such as carbon or silica materials with highvolume ratio and good compatibility can also be usedent bio-label for CL enzyme immunoassay. Zhang andtors investigated a novel CL immunoassay methodultiple enzyme layers assembled multiwall carbon

(MWCNTs) as signal amplication labels employingO2HRPbromophenol blue enhanced CL system [129].y, horseradish peroxidase was assembled onto MWC-tes layer-by-layer through electrostatic interactions.nge from 0.02 to 2.0 ng mL1 was obtained with themit of 8.0 pg mL1 which was 2 orders of magnitudestandard ELISA method. Functionalized mesoporous sil-

rticles (MSN) have gained great interest due to theirperties, including good monodispersity, large surfaceore volume, controlled pore structure, and high ther-echanical stability [130]. Chens group reported an

ly conjugated approaches.


-linke(from Ref. [124

ultra-sensitusing HRP-f[131]. Becathe amounconjugatedtivity. The a0.140 ng m

3.3.2. ElectrElectroc

eration of electron-traproduced w[132]. It comCompared holds the aalso exhibiincluding thECL investi[133135], been applideterminatECL-based research indiagnosis wsensitivity, For immunoso ECL tags such as Ru cdendrimer-Fig. 7. Schematic protocol of the developed sandwich-type CL enzyme

] with permission.)

ive CL immunosensor of carcinoembryonic antigenunctionalized mesoporous silica nanoparticles as labelsuse the large surface area of MSN carriers increasedt of HRP bound per sandwiched immunoreaction, the

provided a much higher signal and increased sensi-nalysis showed a linear response within the range ofL1.

ochemiluminescencehemiluminescence (ECL) technique involves the gen-species at electrode surfaces that then undergonsfer reactions to form excited stated, and light ishen the excited molecule decays to the ground statebines the electrochemical and luminescent techniques.

with the conventional CL, the ECL technique not onlydvantages of sensitivity and wide dynamic range, butts several advantages of the electrochemical methode simplicity, stability, facility. Since the rst detailed

gations described by Hercules and Bard et al. in 1960ECL analysis has received considerable attention anded in many elds, such as environmental pollutantion, pharmaceutical analysis, and immunoassay [136].immunoassays have attracted intensive and extensiveterests due to their important applications in clinicalith the promising advantages, such as simplicity, highreproducibility, rapidity and low background [137,138].assay, most of the biological targets are not ECL-active,

are required to label the biomolecules with ECL reagent,omplex, luminol and its derivatives, quantum dots andencapsulated palladium nanoparticles (Fig. 8) [139].

After ECwas rst reammoniumquickly devapplicationand stabilitNowadays,label and trwidely usebased on ttion of the rpolymerizacarcinoembon the elecside chain omulation oThe coupledsolution. Tha detection

In compsolid-state sive ECL redesign andbeen madetrode surfaelectroactiva series of as electroaHowever, ibecause thed immunoassay by using magnetic beads.L from Tris(2,2-bipyridyl)ruthenium(II) ([Ru(bpy)32+])ported in 1972 [140] in acetonitrile using tetrabutyl-

tetrauoroborate as the electrolyte, Ru(bpy)32+ waseloped as an important ECL emitter with outstandings due to its superior properties including high sensitivityy under moderate conditions in aqueous solution [141].

ECL immunoassay based on the system of Ru(bpy)32+ asipropylamine (TPA) or diketone as coreactant are mostd. Wus group developed a novel ECL immunosensorhe Ru(bpy)32+-TPA ECL system through immobiliza-eductant 2-(diisopropylamino)ethylamine (DPEA) withtion-assisted signal amplication for determination ofryonic antigen [142]. Growth of the polymer materialstrode surface provided numerous epoxy groups on thef poly-glycidyl methacrylate, which allowed the accu-

f ECL coreactant DPEA on polymers acrylamide bonds. DPEA on polymers sensitized the ECL of Ru(bpy)32+ ine proposed immunosensor is extremely sensitive with

limit of 0.5 pg mL1.arison to the solution-phase Ru(bpy)32+ ECL system,Ru(bpy)32+ ECL can reduce the consumption of expen-agent, enhance the ECL signal, simplify experimental

create a regenerable sensor [143]. Great efforts have toward immobilization Ru(bpy)32+ on a solid elec-ce. One of the efcient methods is using Ru(bpy)32+ ase ECL labels immobilized onto the electrode throughrecognition reactions. Typically, Ru(bpy)32+ can actedctive ECL labels in the sandwich-type immunoassay.t is difcult for Ru(bpy)32+ directly label the antibodyre is no functional group on the Ru(bpy)32+ molecule


Fig. 8. Schem y elecnanohybrids.

(from Ref. [139

that can coticles provilabel in ECchemical pered as a gostability anSardesais gprotein canticle as sigthe Stber cle can be uimmunoassFor exampled with gZhangs gromarkers [14on Ru-silicaRu(bpy)32+

immunoassa novel ECLas the label liposome cand avoid tmer, such asurface of nECL immuntionalized Tof human immunosenwith a low

Luminolwell knownaroused sopensive reaHowever, Hand unstabto be menwith the caglucose oxi

olacty [15dditolong

nananc

largeampls a lamun

lumiThusarticlcatioanordy [1ndaratic representation of preparations of tracing tag, and ECL annihilation strategy b

] with permission.)

valently connect with biological substances. Nanopar-de new possibilities for application of Ru(bpy)32+ asL immunoassay because of their special physical androperties. Especially, silica nanoparticles are consid-od matrix because of their biocompatibility, chemicald their surfaces are easy functionalized and modied.roup reported a ECL immunosensor for detection ofcer biomarkers using Ru(bpy)32+ doped silica nanopar-nal amplication [144], which could be prepared bymethod [145147]. The prepared Ru-silica nanoparti-sed as the signal amplication to label proteins in ECLays due to the relatively easy modied silica surface.e, Ru-silica nanoparticle has been respectively modi-old and nanoporous gold nanoparticles by Yuan andups and applied as the labels for determination of cancer8,149]. Thus, stable and sensitive ECL biosensors based

nanoparticles can be successfully prepared. Moreover,

gluconneouslThus, aand pr

Theperformload a signal used aECL imtion of[157]. nanopampligold nantiboof secocan be encapsulated in liposome as the label in ECLay. Bard and Wangs group has, respectively, proposed

immunoassay with Ru(bpy)32+-encapsulated liposome[150,151]. Great signal amplication was achieved sinceould encapsulate large amount of reporter moleculeshe loss of biological activity. A cation-exchange poly-s Naon, can effectively immobilize Ru(bpy)32+ on theanostructure materials. Maos group reported a newosensor based on Ru(bpy)32+ doped-TiO2 (Naon func-iO2) nanoparticles labeling for ultrasensitive detectionchorionic gonadotrophin (HCG) [152]. The proposedsor can perform the ultrasensitive detection of HCG

detection limit of 0.007 mIU mL1. as one of the most efcient ECL reagents is the best. In the ECL system, luminol-H2O2 ECL system has

me concern due to its low oxidation potential, inex-gent consumption and the high emission yields [153].2O2 as coreactant suffered from difculty in labelingle in the detection solution. Fortunately, it is worthtioned that H2O2 is the product of some substratestalysis of corresponding enzymes [154]. For example,dase (GOD) can catalyze the oxidation of glucose to

of luminol, presence ofsignals and8 ng mL1. Ybased on sticles for siwas achievsecondary nanoparticlmodied elproper amoan ECL immnanoparticl[160].

Quantumbeen extenfeatures incphotochemtation specmodicatiolight emissGreat attentrocatalytic reduction toward dissolved O2 at PdNPs@PMM5/SWNH

one in the presence of oxygen, producing H2O2 simulta-5]. Moreover, glucose is stable in the detection solution.ion of glucose instead of H2O2 could improve stability

the lifetime of the immunosensor.ostructures can promote the evolution of high-e ECL immunosensors. They can be used as carriers to

amount of ECL label and thus afford substantial ECLication [156]. Generally, luminol could not be directlybel (except conjugated with gold nanoparticles) in theosensor of luminol due to the fact that the functionaliza-nol has lower CL efciencies than the parent compounds, there are growing considerable interest in employinges as carriers for the immobilization of GOD for signaln. Xu et al. reported a cathodic ECL of luminol based on

ods multilabeled with glucose oxidase and secondary58]. The gold nanorods were not only used as carriersy antibody and GOD but also catalyzed the ECL reaction

which further amplied the ECL signal for luminol in the

glucose and oxygen. A linear relationship between ECL the PSA was obtained in the range from 10 pg mL1 touans group developed an ECL immunoassay of luminol

ynergetic catalysis effect of enzyme and Pd nanopar-gnal amplication [159]. Greatly enhanced ECL signaled by using bioconjugates featuring GOD labels andantibodies linked to functional carbon nanotubes-Pdes, which exhibit attractive catalysis activity when theectrode was detected in the working buffer containingunts of glucose. Moreover, their group also proposedunosensor based on glucose oxidase supported on Aues decorated multi-walled carbon nanotubes as labels

dots (QDs), as a new kind of ECL luminophores, havesively studied due to their numerous advantageousluding high quantum yield, low photobeaching, highical stability, size-tunable emission and broad exci-tra for multicolor imaging, and feasibility for surfacen [161]. Bards group found that Si could generateion during potential cycling or pulsing (ECL) [162].tions have been paid to applying QDs as ECL labels for


bioassays. Particularly, the modied QDs have been successfullyused as ECL labels in immunoassays.

Most of the ECL immunoassays of QDs are based on the quench-ing, inhibition, or enhancement of the ECL intensities via the wellstudied coras the coreoxidation owith an ECthe luminoused systemdeveloped trode and ureported a human chofunctionalizcation [16of CdTe QDdetection wQians groutum dots cdetection othe high loIgG detectiusing silicaNear-Infrarsilica nanosof biomarkethe coreactsensitivity f

QDs ECLECL immunimmunoasstein antigenquenching factivated Ccomposite [related to th10 pg mL1

gated an EC(AFP) basedECL of grapECL intensitration in thof 20 ag mL

CdS nanocrultrasensitivia an efcby sandwic1.0 fg mL1.

3.3.3. FluorFluoresc

dominant arescence imin the eldof uorescesay. Nanopimmunosencal, electrobeen recognals, which pbiotechniquon the -Nachieved fogoat anti-hu

Semiconductor nanocrystals (quantum dots, QDs) haveattracted a great deal of attention due to some unique prop-erties such as size-controlled uorescence, high uorescencequantum yields, and stability against photobleaching. QDs were

nalizns [1CdTeve p/ZnSin dnS Qimat

ased n (PSg of

sens samed ad a ln [17sor, has atd frorategsor dcentoparing

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sens biol

has to iing td na

the eactant ECL systems which took S2O82, H2O2, SO32

actants [163]. The coreactant is a species that, uponr reduction, produces an intermediate that can reactL luminophore to produce excited states. CdTe QDs asphor and K2S2O8 as the coreactant is the most widely

in sandwich enhanced ECL immunoassays. Lis groupan ECL immunoassay at a nanoporous gold leaf elec-sing CdTe quantum dots as labels [164]. Zhangs groupnovel ECL immunosensor for sensitive detection of

rionic gonadotrophin antigen (HCG-Ag) using CdTe QDsed nanoporous PtRu alloys as labels for signal ampli-5]. Due to signal amplication from the high loadings, 4.67-fold enhancements in ECL signal for HCG-Agas achieved compared to the unamplied methods.

p proposed a versatile immunosensor using CdTe quan-oated silica nanosphere as a label for ultrasensitivef a biomarker [166]. Due to signal amplication fromading of CdTe QDs, 6.6-fold enhancements in ECL foron were achieved compared to the method without

nanosphere as the carrier. Wangs group reported aed ECL immunosensor by using CdTe/CdS QDs taggedpheres as signal amplication for sensitive detectionr [167]. All of the reported works basing on K2S2O8 asant provided an effective way with good stability andor protein detection.

quenching principles were also employed in sandwichoassay for protein analysis. Tians group reported an ECLay method for ultrasensitive detection of prostate pro-, by remarkably efcient energy-transfer induced ECLrom the CdS QDs sensitized TiO2 nanotube array to thedTe QDs functionalized multi-wall carbon nanotubed168]. The ECL intensity decrement was logarithmicallye concentration of the PSA in the range of 1.0 fg mL1 towith a detection limit of 1 fg mL1. Guos group investi-L immunosensor for determination of alpha fetoprotein

on the label CdSe/ZnS QDs effectively scavenging thehene-CdS QDs-alginate composite [169]. The quenchedty depended linearly on the logarithm for AFP concen-e range from 0.05 to 500 fg mL1 with a detection limit1. Songs group reported an ECL immunoassay based onystals functionalized TiO2 nanotube arrays [170]. Theve rabbit IgG detection is achieved on the compositesient ECL quenching process by CdTe QDs introducedhed immunoreaction, and shows a detection limit of

escenceence is by far the method most often applied and is thenalytical approach in a large variety of schemes. Fluo-munoassay, as one of the most common approaches

of optical biosensors, combines the high sensitivitynce detection with the high selectivity of immunoas-article-based signal amplication of sandwich-typesors has attracted wide interest due to its unique opti-

nic, and biocompatible properties. -NaYF4:Yb,Er hasized as one of the most efcient luminescent materi-roves to be an ideal choice for biolabeling in variouses [171]. Lius group reported a immunoassay basedaYF4:Yb,Er nanophosphors [172]. High sensitivity isr the proposed immunoassay and as low as 0.1 ng mL1

man immunoglobulin G can be detected.

functioantigeusing sensitia CdSealbumCdSe/ZapproxQDs-bantigeimagina veryserumproposdots anplasmibiosenand hderivestep stbiosenthe con

Nanquenchcially, quenchand resity oruoroiresceinanti-immunnanopsay bananoption ofwith thdoes nand m

3.3.4. SPR

cal couof SPRhave avariouitoringwith imcially ftheir hthe uslimitedresult the binthe devhighertion ofweighturgentextend

Golshift ined with antibodies and used as uorescent probes for73]. Cuis group developed a versatile immunoassay

QDs as electrochemical and uorescent labels forrotein detection [174]. Tu and his workers developed

QDs-based optical immunosensor for human serumetection (Fig. 9) [175]. The detection limit for theD-based immunosensor developed in this study wasely 3.2 105 mg mL1. Kermans group developed aimmunosensor for the detection of prostate-specicA) using uorescence microscopy [176]. Fluorescencethe substrate surface illuminated the QDs, and provideditive tool for the detection of PSA in undiluted humanples with a detection limit of 0.25 ng mL1. Lis group

portable uorescence biosensor based on quantumateral ow test trip (LFTS) for detection nitrated cerulo-7]. A lateral ow test trip, also called a dry-reagent stripas been becoming a powerful tool for protein analysistracted increasing attention. Due to the advantagesm QDs and LFTS, a rapid, sensitive, selective, and one-y has been developed. And the portable uorescenceisplays rapid responses for nitrated ceruloplasmin withration as low as 1.0 ng mL1.ticles possessing the ability of efcient uorescencehave been hold for great promised for biosensors. Espe-nanoparticles have a very strong ability for uorescentbecause they can monitor receptor or ligand binding

events through changes in the uorescence inten-ime [178]. Cui and his co-workers have developed anoassay based on the uorescence quenching of uo-hiocyanate caused by gold nanoparticles coated withprotein monoclonal antibody, where the sandwich-type

plex was separated by a magnetic eld using magnetices [179]. Yus group reported a similar immunoas-n the uorescence quenching of uorescein by goldes coated with antibody dissociated by the mixed solu-ium hydroxide and trisodium citrate [180]. Comparedthod provided by Cui and co-workers [174], the methoded the complicated preparation of the magnetic beadstic nanoparticles coated with antibody.

ce plasmon resonance (SPR)e kind of physical optics phenomena produced by opti-g of thin metal lms [181]. Since the rst applicationnology for a biosensor in 1983 [182], SPR biosensorsted increasing attention and have been widely used inds including medical diagnostics, environmental mon-d safety, and security [183]. SPR technique combiningnosensors have recently attracted a lot of attention espe-rect detection of biomolecular interactions because ofnsitivity and real-time monitoring [184,185]. However,he SPR method is hindered and the system displayedsitivities when the change of the refractive index as ading process is often small, which might be caused by

of small molecular weight materials [186,187]. Withment of life science, there is a growing requirement foritive detection technologies in analysis and the detec-ogical samples with low concentration and molecular

attached more and more importance [188]. Thus, it ismprove the sensitivity of the SPR immunosensors forhe range of application of the tool.noparticle has been known to provoke an outstandingangle of plasmon resonance, and it combined sandwich


(from Ref. [175

assay are thsensitivity oSPR immunenhanced a[189]. In thC4 had gooand the lowby the direface plasmoantigen (PSFor the detticle complwith femto-immunosenprostate-spamplicatioFig. 9. Schematics and real picture of a QD-based optical biosensing system, includin] with permission.)

e techniques most commonly applied for enhancing thef SPR immunosensor. Lius group reported an enhancedosensor based on the sandwich assay and colloidal-Au-ssay for determining the concentration of human C4e colloidal-Au-enhanced sandwich assay, the humand response in the concentration range 0.055 g mL1

est concentration is 40-fold lower than that obtainedct assay. Chois group developed a ultra-sensitive sur-n resonance based immunosensor for prostate-specicA) using gold nanoparticle-antibody complex [187].ection of PSA, the use of the proposed Au nanopar-ex enabled 103-fold signal enhancement in parallellevel detection. Uludags group reported a point-of-caresor for the detection of the cancer biomarkers (totalecic antigen, tPSA) using SPR sensors with gold signaln [190]. Gold nanocomposites are also excellent agents

functioned immunosena sandwichAFP secondsignicant the used of er. The eximmunosenfor AFP dete

3.4. Multia

Multianare measuin clinical tional singg diode laser, lens, long-pass lter, and photodiode.

as an amplier to increase the sensitivity of the SPRsor. Liangs group reported a novel SPR sensor based on

immunoassay to detect AFP by employing Fe3O4@Au-ary antibody conjugates as the amplication reagent. Aincrease in sensitivity was therefore afforded throughFe3O4@Au-secondary antibody conjugates as an ampli-perimental results demonstrate that the designed SPRsor possessed a good sensitivity and a high selectivityction [183].

nalyte immunoassays

alyte immunoassay, in which two or more analytesred simultaneously in a single assay, is requestedlaboratories [191,192]. Compared with the tradi-

le-analyte immunoassay, multiplexed immunoassay


ells an(from Ref. [201

with the addecreased scost as comanalytical mcessful deveconcern is lyte from thpresent meresolved asarrays withventional mfor electrocsandwich-ttrochemicaarray basedenriched golabels [187immunoassusing gold ratio of HRindividuallyferent analAlthough mbiological rminiaturizefunctional plenge. Hencredox tags, multiplexed

An alternformed by advantage isufcient sbetween n

sepoass07].

elecltiplgnalFig. 10. Schematic diagrams of immunosensors array with 4 12 c] with permission.)

vantages of shortened analysis time, simplied signals,ampling volume, improved test efciency, and reducedpared to parallel single-analyte assays is a promisingethod in sample analysis [92,192,193]. For the suc-lopment of multiplexed immunoassays, the rst majorhow to discriminate each signal for one special ana-

spatialimmuntalk [2on thefor muious sie multiple antigenantibody reactions. Nowadays, thethods are mainly based on the multilabel and spatiallysay protocol [194198]. Parallel ultra-microelectrode

small inter-electrode distances behave similarly to con-acroscopic electrodes on the typical time-scales used

hemical immunosensors [199]. Jus group reported twoype multiplexed immunoassays for simultaneous elec-l detection of multiple biomarkers on an immunosensor

on various nanostructure labels, e.g. carbon nanotube-ld nanoparticles as the labels [200], or gold nanoparticle]. Zongs group reported a chemiluminescence imageay of multiple tumor markers fro cancer screening bynanoparticle-based bioconjugated with a high molarP as detection antibodies (Fig. 10) [201]. Arrays of

addressable electrodes can be used to determine dif-ytes or to provide spatially resolved measurements.odication of the individual electrodes with differentecognition elements would enable the construction ofd immunosensor arrays, the directed immobilization ofroteins on individual microscopic regions is still a chal-e, multilabel methods based on enzymes, metal ions,and quantum dots, have been used for development of

immunosensings and immunoassays [201205].ate strategy for multiplexed immunoassay can be per-use of a single-enzyme label [206], which offers ann simplicity as compared to multiple labels but requireseparation to prevent signal interference (cross-talk)eighboring electrodes. Wilson has described enough

been develthe most pticles used loading a lwich immuelectrochemon enzymedriven accimmunoassers using mas the labethese methdetection wis critical anthis issue, the approaenzyme adsstructure wenzyme moenzyme actthe enzyme

Magnetohave becomtiple biomo[72,211,212ing througimmunoassbeen fabricad CL imaging immunoassay procedure.

aration of electrodes which can perform individualay of multiple proteins without amperometric cross-Jus group immobilized an electron-transfer mediatortrode to avoid cross-talk when a single-enzyme labelexed immunoassay of proteins was used [208]. Var-

amplication technologies using nanomaterials have

oped for ultrasensitive detection of proteins. One ofopular strategies is enzyme-functionalized nanopar-as tracers to enhance the sensitivity of detection byarge amount of enzymes toward an individual sand-nological reaction event. Du reported a multiplexedical immunoassay of phosphorylated proteins based

-functionalized gold nanorod labels and electric eld-eleration [194]. Jeon developed a new gravimetricay for sensitive detection of multiple protein biomark-ultifunctional hybrid nanoparticles (Fe3O4/TiO2/SiO2)ls on silicon microcantilever arrays [209]. However,ods usually employed conductive nanomaterials for theith no serious consideration of enzyme stability, whichd important for the delity of ELISA signals. To tackle

Kim reported a multiplexed immunoassay based onch of nanoscale enzyme reactors (NERs), in which theorption into mesoporous silica with the bottleneck poreas followed by the chemical crosslinking of adsorbedlecules [210]. The NER approach could stabilize theivity and maintain high enzyme loading by preventing

leaching via a ship-in-a-bottle effect.-controlled molecular electronics and bioelectronicse new tools for simultaneous monitoring of mul-lecules in food, environmental and clinical samples]. Magnetic sorting protein assay systems with vary-hputs have been built and employed for multipleays [213,214]. Batch-type magnetic separators haveted on a single chip for trapping and directed sequential


elution of magnetic particles in owing uids [215217]. Tangreported a new ow-through multiplexed immunoassay proto-col for simultaneous electrochemical determination of CEA andAFP in biological uids using biofunctionalized magnetic graphenenanosheets as immunosensing probe and multifunctional nanogoldhollow microspheres as distinguishable signal tags [218]. The assaywas based on the catalytic reduction of H2O2 at the various peakpotentials in the presence of the corresponding mediators.

Quantum dots (QDs) have shown great potential in the mul-tiplexed immunoassays because of their unique advantages:nanoscale size similar to proteins, broad excitation spectra formulticolor imaging, robust, narrowband emission, and versatil-ity in surface modication [174,219221]. Compared with organicuoropores, QD provide tunable, symmetrical and narrow emis-sion bands with high photostability. Cooper and co-workerreported upon the application of quantum dot barcodes pre-pared by layer-by-layer biological self-assembly of quantumdot-biotin and quantum dot-streptavidin on magnetic beads forqualitative multiplexed immunoassays [222]. More signicantly,different-sized QDs can be excited with a single wavelengthexcitation light. Therefore, QD are considered one of the mostpromising materials in the multiplexed immunoassays. Zhangdeveloped a new cross-talk-free duplex uoroimmunoassay forcancer-related biomarkers based on dual-color quantum dotsas detection elements [223]. Zeng and colleague constructeda multiple homogeneous immunoassay based on a quantumdots-gold nanorod FRET nanoplatform by using green QDs andred QDs as distinguishable signal tags [224]. More signicantly,multicolor quantum dots could be also used as the labels formultiplexed uorescence polarization immunoassay [225]. In themultiplexed electrochemical immunoassays, the coding bioassayrelies on the use of different inorganic-colloid nanocrystal tracers,whose metvoltammetrchemical impathogens utals as disti

printed electrode [226]. Kong et al. developed an electrochemicalimmunoassay for simultaneous determination two tumor mark-ers using CdS/DNA and PbS/DNA nanochains as distinguishabletags [227]. Due to the difference in the oxidation potentials ofthese metal components, they exhibited sharp and well resolvedstripping voltammetric peaks at various peak potentials. Further,some metal ions comprising cadmium, copper and zinc usu-ally display various voltammetric characteristics at the differentapplied potentials. Favorably, these metal ions can be strippedfrom the corresponding metal nanoparticles under the harsh con-ditions. Zhus group designed a new multianalyte electrochemicalimmunoassay for detection of two human cardiopathy biomark-ers using metal-ions (i.e. Cd2+ and Zn2+) functionalized titaniumphosphate nanospheres as distinguishable signal tags [228]. Unfor-tunately, the QDs-based electrochemical detection often requiresharsh detection conditions, including nanocrystals dissolution,high potential accumulation, and deoxygenation, which is not suit-able for clinical application.

An alternative approach is to utilize enzyme-catalyzed metalion deposition for the amplication of detectable signal. Comparedwith AuNPs, silver nanoparticles (AgNPs) can be oxidized at morenegative potential with a relatively sharp peak, which is favorableto obviating the interference of reducing species and improvingthe detection precision and sensitivity [229]. The silver deposi-tion can be carried out with the aid of enzyme, nanoparticles orother reduction agent. Lai and co-worker reported several meth-ods for electrochemical stripping analysis of silver nanoparticles inthe sandwich-type multiplexed immunoassays by coupling differ-ent silver deposition techniques and nanoparticles labels, e.g. silvernanoparticles catalytically deposited by gold nanoparticles andenzymatic reaction [230], nanogold label-induced silver deposition[231], and silver-nanoparticle-enriched carbon nanotube labels-

d silarticling werhere

Table 1Comparison of s for t

Method ng mL

Electrochem Amperomet 1 Electrochem Electrochem 06

ElectrochemFluorescenceFluorescenceElectrochemElectrochemElectrochem 2 Electrochem Electrochem ElectrochemElectrochem Electrochem 5 Chemilumin Electrochem Magnetic imELISA ElectrochemChemiluminElectrochem Electrochem 4 ElectrochemElectrochem ElectrochemElectrochem

a GOx: glucoal components yield well-resolved, sensitive strippingic signals. Viswanathans group devised an electro-munosensor for multiplexed detection of food-bornesing three metal sulde (CdS, PbS, and CuS) nanocrys-

nguishable tags on a carbon nanotube-modied screen

inducenanopsay uswhich nanosp

analytical properties of various sandwich-type immunosensors and immunoassay

Linear range (ng mL1) LOD (

iluminescence 0.0180 0.0033ric immunosensor 0.01200 0.0001ical immunoassay 0.1100 0.0033iluminescence 5.0 1065.0 104 2.0 1ical immunoassay 0.110 0.01

immunoassay 11000 0.5 immuosensor 0.1100 0.04 ical immunoassay 0.5160 0.08 ical immunosensor 0.001100 0.001 iluminescence 0.00050.5 0.0001ical immunosensor 0.00150 0.0001iluminescence 0.00110 0.0008ical immunosensor 0.00550 0.002 iluminescence 0.055000 0.0024ical immunosensor 0.00010.002 0.0000escence 0.0050.5 0.0041ical immunosensor 0.02120 0.0012munoassay 0.250 0.058

3.060 1.5 ical immunoassay 0.093 escence 0.140 0.04 ical immunosensor 0.00520 0.0017ical immunosensor 0.00011.0 0.0000ical immunosensor 0.00550 0.001 ical immunosensor 0.0052.0 0.0032ical immunosensor 0.180 0.03 ical immunosensor 0.0112 0.005

se oxidase; HRP: horseradish peroxidase; CNT: carbon nanotube.ver deposition [232]. Just as these advantages of silveres, Woo et al. constructed a multiplexed immunoas-uorescent-surface enhance Raman spectroscopic dots,e composed of silver nanoparticle-embedded silicas, organic Raman tagging materials, and uorescent

he detection of CEA using different nanoparticle labels.

1) Labelsa Ref.

Nano-ZnO + GOx [239]AuPt nanochain + HRP [240]CdS/DNA nanochains [227]QD labels [241]B-Galactosidase [242]Europium chelates [243]Without labels [244]Without labels [245]Pt hollow spheres [28]Nano-Au [246]Polyaniline nanobers [247]Ru-Silica-Au composite [149]Nano-Au [248]Carbon nanotubes [249]Gold nanoparticles [250]Gold nanoparticles [251]Hollow Pt nanoparticles [252]PbS nanoparticles [253]HRP without nanolabels [254]Silver-carbon nanotube [232]HRP-SiO2 [131]Pt-HRP [255]Carbon nanotube-HRP [256]AuAg-GOx [155]GOx-SiO2 [257]Fe3O4-Au [258]Au-SiO2-CNT [259]


dyes [233]. Brady and colleague presented a strategy for thesynthesis of multiplexed spectral encoder beads based on combi-nation of different surface enhanced Raman signatures generatedby dye-functionalized silver nanoparticle tags [234]. For detec-tion purpoencoder wication of muof availableencoder beamore ubiqusmall specanalytes.

In additsandwich-tof detectabimmunochrimmunosorend, to furtanalytical psays with ausing carciThe resultsMurray, foreditorial seof analytica[260].

4. Conclud

Inherenttinue to bsandwich-thave descriof detectaband immunlabel-free bleading roleof interfacinal amplibiofunctionopment of properties with nanoping aspectsnumerous chemistry, eDue to diffproperties, to achieve aand to conachieve a beimmunosenbe made wimprove thfor a varietmicro-/nanthroughputvariety of itype immuwill becomlabeling readvantagesassay approit cannot bmultivalent

Acknowledgments

This work was nancially supported by the National NaturalScience Foundation of China (41176079, 21075019), the National

Basicrogrtionae Pron Unaken

nces

Wang7149. Wu, Hat. Bio. Mungusling. Ritte012)

. Qin, Wd. 51 (. Tang. Tang. TangQuint

Gabil1446. Tang. Li, Lterfac. Su, D.. Zhan723.Tang, . Liu, Y

Brakm. Vicend. 44 (. Am5716. Mar. Hock. DOraTothil. Lupp. Jiang. Zhan011) . Li, D.5315. Sun, Zhou,012)

Zeng,. Zhan. Chin,

Kurki. Herv

Yan, H. Diaz005) Tang,8 (201. Shid3213. Yan,. ttp://d. Shan0711. Cai, Yiosens

Feng,. Nam0112Zhang. Dai, FWu, FWu, Yses, the use of these nanoparticle assemblies as anll allow for the straightforward simultaneous identi-ltiple analytes, limited in principle only by the number

unique molecular dye signatures. These multiplexedds are envisioned to have applications that supplementitous uorescence-based bioassay schemes that requiretral bandwidths and large numbers of multiplexed

ion, nanoparticle labels can be used in the otherype multiplexed immunoassays for the amplicationle signal, e.g. colorimetric immunoassay [235,236],omatographic strips [237], and thermally addressedbent assay [238]. As the discussion is coming to theher elucidate the advantages of nanoparticle labels, theroperties of selected immunosensors and immunoas-nd without nanoparticle labels are also compared bynoembryonic antigen (CEA), as an example (Table 1).

strongly highlighted one sentence made by Royce W.mer Editor of the journal of Analytical Chemistry, in anries: Nanoparticles will be part of the heart and soull chemistry and science at large, far off into the future

ing remarks

sensitivity, simplicity, speed, and cost benets con-e strong driving forces for the development ofype immunosensings and immunoassays. Herein webed a variety of nanoparticle labels for the amplicationle signal in different sandwich-type immunosensorsoassays. Despite historic achievements in the elds ofioassays, labeling techniques will continue to play a

in this eld. Nanoparticle labels offer very elegant waysng biomolecule recognition events with inherent sig-cation. Along with developing of labeling techniques,alized nanoparticles have paved the way for the devel-highly sensitive diagnosis devices because of unusualof nanoparticles. Especially, coupling enzyme labelsarticle labels is one of the most exciting and challeng-

of this eld. Success will play a vital role in advancingscientic disciplines, including biomedicine, biology,nvironment science, toxicology, and materials science.erent shapes and sizes probably possessing differentdesigning and developing new strategies are necessary

better understanding of the nanofabrication process,trol particle aggregation and surface interactions. Totter understanding of nanolabels-based sandwich-typesings and immunoassays, great efforts will need toorldwide to design and develop new strategies thate properties of nanolabels. In addition, developing usesy of nanomaterials coupled with enzyme labels ando-uidic devices will offer advanced miniaturized high-

and cost-effective multiplex assays for tagging of a widemportant chemical and biological targets. Sandwich-nosensors and immunoassays exploiting nanoparticlese a common technology increasingly accepted as

agents in

sandwich-type immunosensors and immunoassays exploiting nanostructure labels: a review

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