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Analytica Chimica Acta 690 (2011) 10–25

Contents lists available at ScienceDirect

Analytica Chimica Acta

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anoparticle-based electrochemical detection in conventional and miniaturizedystems and their bioanalytical applications: A review

eena Siangproha, Wijitar Dungchaib, Poomrat Rattanaratc, Orawon Chailapakulc,d,∗

Department of Chemistry, Faculty of Science, Srinakharinwirot University, Sukumvit 23 Rd., Wattana, Bangkok 10110, ThailandDepartment of Chemistry, Faculty of Science, King Mongkut’s University of Technology Thonburi, 91 Prachauthit Road, Bangmod Thung Kharu District, Bangkok 10140, ThailandDepartment of Chemistry, Faculty of Science, Chulalongkorn University, Patumwan, Bangkok 10330, ThailandNational Center of Excellence for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand

r t i c l e i n f o

rticle history:eceived 26 October 2010eceived in revised form 26 January 2011ccepted 27 January 2011

a b s t r a c t

With recent advances in nanotechnology making more easily available the novel chemical and physicalproperties of metal nanoparticles (NPs), these have become extremely suitable for creating new sensingassays. Many kinds of NPs, including metal, metal-oxide, semiconductor and even composite-metal NPs,have been used for constructing electrochemical sensors. This article reviews the progress of NP-based

vailable online 26 February 2011

eywords:etal nanoparticles

lectrochemical Sensorsiniaturized Systems

electrochemical detection in recent applications, especially in bioanalysis, and summarizes the mainfunctions of NPs in conventional and miniaturized systems. All references cited here generally show one ormore of the following characteristics: a low detection limit, good signal amplification and simultaneous-detection capabilities.

© 2011 Elsevier B.V. All rights reserved.

Wijitar Dungchai, Ph.D. is a lecturer of Departmentof Chemistry, Faculty of Science, King Mongkut’s Uni-versity of Technology Thonburi. She received her B.S.degree and Ph.D. in chemistry from Chulalongkorn.Her research involves the development of optical andelectrochemical devices using lab-on-paper platformsfor medical diagnostics.

mmunosensorsiosensors

Weena Siangproh, Ph.D. is a lecturer of Department ofChemistry, Faculty of Science, Srinakharinwirot Uni-versity. She received her B.S. degree in chemistryfrom Srinakharinwirot University and Ph.D. in chem-istry from Chulalongkorn University. She received theexcellent thesis award from the Chulalongkorn Uni-versity and Doctoral thesis award from the NationalResearch Council of Thailand in 2007, respectively.Her current research interests include electroanalyt-ical applications, flow-based system and lab-on-chipdevices for quantitative drug, biological compounds

and diagnostics.

∗ Corresponding author at: Department of Chemistry, Faculty of Science, Chulalongkorel.: +66 2 218 7615; fax: +66 2 234 1309.

E-mail address: corawon@chula.ac.th (O. Chailapakul).

003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2011.01.054

n University, Patumwan, Bangkok 10330, Thailand.

W. Siangproh et al. / Analytica Chimica Acta 690 (2011) 10–25 11

Poomrat Rattanarat is a Ph.D. student in Departmentof Chemistry, Faculty of Science, Chulalonkorn Uni-versity. He obtained B.S. degree in chemistry fromSrinakharinwirot University in 2008. His researchinterests include the development of immunosensingand nanoparticles into microfluidic and lab-on-paperplatforms for various applications.

Orawon Chailapakul, Ph.D. is the Associate Profes-sor of Department of Chemistry and a leader of theElectrochemical Research Group at ChulalongkornUniversity. She received her B.S. and M.S. degreesin chemistry from Mahidol University and Chula-longkorn University, respectively. She received herPh.D. degree from the University of New Mexico,USA. Her current research interests include electroan-alytical detection, flow-based system and lab-on-chipdevices for various applications, immunoassay, andbattery. In 2006 and 2008, she received the BestArticle Award and JAFIA Scientific Award from theJapanese Association for Flow Injection Analysis

(JAFIA), respectively. In 2009, she received TRF-CHE-Scopus Researcher Awardsfrom the Thailand Research Fund, CHE, and Elsevier publisher.

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. Introduction

Recently, nanoparticles (NPs) of different compositions andimensions have become used as versatile and sensitive trac-rs [1]. The creation of designer NPs for enhanced sensitivity inensing applications greatly benefits from their small size, whereheir properties are strongly influenced by increasing their sur-ace area [2–7]. Indeed designer NPs are one of the most excitingreas in modern electroanalytical analysis because they offer excel-ent prospects for creating highly sensitive and selective assays.lectrochemical sensing methods based on the modification of par-icular metal NPs on conductive substrates is a fascinating anduickly expanding research area [8–12]. The remarkable electro-hemical features of metal NP-modified electrodes are now widelymployed in conventional systems. To reduce the use of reagentsnd the required electroanalysis time, a miniaturized system haseen introduced. In this manuscript, the metal-NP electrochemicalensors used for electroanalytical purposes, especially, in bioap-lications are reviewed. Biochemical substances are one of thessential companions of life on earth, it has been central to thevolution of human civilizations in healthy. Therefore, even sincehe discovery of metal-NP, researchers have been exploring theirotential in bioapplications, which made these applications arehe most expanded in recent years. One of the reasons to focusn the use of metal-NP based electrochemical detection in bio-nalysis may be concern the importance of the diagnosis andrevention of diseases. Indeed, metal-NPs have many interestingnd unique properties potentially useful in a variety of biologicalnd biomedical systems and devices. Such the mentioned inspi-ations, the electrochemical characteristics of metal NPs and theensing applications derived from these materials are discussed,nd their advantages and weaknesses are explored. We envisage aromising future for the use of these metal-NP-modified electrodes

n both conventional and miniaturized systems.

. Metal-NP-based electrochemical detection inonventional systems

Research into metal-NP-based electrochemical detection forioanalysis has drawn increasing attention from scientists in recentears owing to their extreme usefulness. Many types of metal NPsith different sizes and compositions are now available, facili-

ating their use in electrochemical applications as both chemical

ensors and biosensors. These metal-NPs are of high surface area,igh mechanical strength but ultra-light weight, rich electronicroperties, and excellent chemical and thermal stability [13]. Thenique properties of nanoscale materials led to very high sensi-ivity that is a gold purpose in biological assays. Currently, highly

sensitive methodologies are needed for measuring disease diagno-sis markers present at ultralow levels during early stages of diseaseprogress. The emergence of metal-NPs is not only opening newscopes for highly sensitive bioaffinity and biocatalytic assays fornovel biosensor and chemical sensors protocols but also leads tothe excellent prospects for designing highly selective bioassays ofvarious applications such as nucleic acids and proteins [2]. Voltam-metric techniques are certainly the most widely employed methodsin electrochemical analysis, including sensing applications basedon metal, metal-oxide, semiconductor and composite metal NPs. Inthe subsequent sections, several examples of the use of voltammet-ric methods with NP detectors are described with the content beingcategorized according to the nanomaterial type. In the final partof this section, we give an overview of the other electrochemicalmethods that use metal-NP-based constructs as sensors.

This review concentrates on AuNPs due to their superb proper-ties. AuNPs have been known since ancient times as colloid gold andemployed in many applications. AuNPs can be synthesized in a var-ious forms and sizes using different chemical methods. Typically,precious metals, including not only Au, but also Pt, Pd and Ag, areresistant to oxidation reactions and so they are selected to be theworking electrode in electrochemical systems. Au or AuNPs are themost frequently chosen of the precious metals for electrochemicalsystems due to the relatively low cost and ease of preparation. TheAuNPs can be stabilized by negatively charged anions around thesurface making it easily modified by a wide range of biomoleculesbearing a thiol moiety. The use of AuNP to enhance detection sen-sitivity is reviewed as follows.

2.1. The use of AuNPs in voltammetry for the detection of smallbiomolecules

AuNPs have been widely used and have attracted considerableattention in analytical and biomedical areas due to their speed andease of use in chemical synthesis, their narrow size distribution andtheir convenient labeling of biomolecules [14].

The preparation of electrochemical sensors using AuNPs coatedon a variety of electrodes for the determination of small moleculeshas been performed, including with AuNP-modified glassy car-bon electrodes (GCE) for the detection of dopamine (DA) anduric acid (UA) in the �M range [15]. Fig. 1 illustrates the elec-trochemical behaviour of DA, AA and UA at bare and modifiedelectrode. It is noticed that the overlapped voltammetric peak is

resolved into three well-defined peak by using the AuNP-modified.Therefore, NPs offer the benefit for simultaneous determination. Inaddition, various shapes of gold, such as gold nanorods [16] andgold-nanoclusters [17], can be used as a template for the detec-tion of DA, leading to detection thresholds within the nM range. A

12 W. Siangproh et al. / Analytica Chim

Fig. 1. Cyclic voltammograms of a bare GCE (1) and GNP/Ch/GCE (2) in 1/15 PBS (pH7rF

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.0) containing 0.05 mmol L−1 AA, 0.01 mmol L−1 DA and 0.01 mmol L−1 UA. Scanate: 50 mV s−1.rom Ref. [15], with permission.

anocomposite composed of poly(3-methylthiophene) and AuNPsas proposed for the sensitive determination of DA and UA in theresence of ascorbic acid (AA) [18], but it was not as sensitives the nanocluster modified electrodes [16,17]. The ultrasensi-ive and ultraselective electrochemical sensor for the detectionf nanomolar concentrations of DA in the presence of AA cane fabricated from a multi-walled carbon nanotube (MWCNT)-rafted silica network (silica NW) and AuNPs (MWCNT-g-silicaW/AuNPs) [19]. When incorporated with indium tin oxide (ITO)s an ITO/MWCNT-g-silica-NW/AuNP-modified electrode, a wideinear current response for DA was obtained with a detection limitf 0.1 nM, the lowest detection limit reported for DA.

Self-assembly is one of the most common methods used to pre-are AuNPs on the electrode surface [20]. For example, cysteaminean be used as the binder between AuNPs and the GCE surfaceo determine the DA level in the presence of a high concentra-ion of AA. Similarly, the immobilized AuNPs in a binary mixedelf-assembled monolayer onto a gold surface can be employed forhe electrochemical analysis of DA and AA together in an aque-us media in the �M detection limit range [21]. However, theinear detection ranges and detection limit obtained for both ana-ytes were higher than those reported with the AuNP-GCE reportedbove [20]. In addition, a three-dimensional l-cysteine monolayerssembled on AuNPs on gold and GCEs was reported for the simul-aneous determination of UA and AA [22]. Similar to the previously

entioned work, this modified electrode could separate UA and AAn the low �M detection limit range. However, despite the relativen sensitivity, against this a large concentration range for detection

as obtained using l-cysteine/AuNPs on the GCE.Furthermore, a boron-doped diamond (BDD) electrode was

valuated as the novel electrode support for the modificationf AuNPs to selectively detect DA in the copresence of AA [23].ompared with Raoof’s work [21], the detection limit of DA wasecreased ∼100-fold, which may be the result of the unique prop-rties of the BDD electrode in terms of a low background current.

1-Pyrene butyric acid-functionalized graphene (PFG) sheetsere proposed for AuNPs to self-assembly of AuNPs onto its sur-

ace to form a sensor for the detection of UA [24]. A GCE modifiedith AuNPs and the PFG composite showed a rapid response, high

ensitivity (20 �M upwards), strong electrocatalytic activity and aigh electrochemical stability.

The use of self-assembly for the preparation of AuNP electrodeso detect the electrochemical properties of epinephrine (EP) by

ica Acta 690 (2011) 10–25

voltammetry has been described [25]. This electrode can be usedfor solving the traditional problem of interference from AA due totheir overlapping peaks. There are also two other reports by Luczak[26,27] that evaluate the ability of modified AuNPs on a gold elec-trode to detect EP in the presence of UA and AA as the interferingmolecules. These two papers report very similar procedures forelectrode preparation, with just some slight modifications in thebinder type (thiodiprionic acid (TDPA) and 3-mercaptopropionicacid (MPA)) and modification method. The other interesting workfor EP detection is fabricated by the electrochemical deposition of agold nanocluster on an ultrathin overoxidized polypyrrole (PPyox)film, forming a nano-Au/PPyox composite on the GCE [28].

Besides the biomolecules mentioned above, AuNPs have beenused as sensors for the detection of homocysteine at the nM scalewith a carbon paste electrode (CPE) [29], cytochrome c at thepM to nM level [30], tryptophan with a CNT/GCE at the nM scale[31], bilirubin with MWCNT–GCE at 120 nM upwards [32] andhemoglobin (Hb) with PASE–MWCNT at the low nM level [33].Multianalyte detection by electrochemical biosensors based onaptamer and nanoparticle-integrated bio-barcode has been devel-oped, in this case for detection of adenosine and thrombin at thelow pM level [34].

These modified electrodes display satisfactory results comparedto standard analytical techniques, with a marked enhancementin the current response, yielding a sensor with an excellent elec-trocatalytic response, fast response time, long-term stability andreproducibility. Table 1 summarizes the recent reports on the useof metal NPs to improve the sensitivity for the detection of smallbiomolecules.

2.2. The use of AuNPs in voltammetry for DNA detection

Currently, there are numerous papers reporting on the use ofAuNPs in the design of electrochemical biosensors. This reviewdeals with articles related to the use of nanomaterials as sen-sors for DNA detection, in which AuNPs are the current favoritematerial and are reported to be able to play an important role inDNA detection [35]. Although many kinds of NPs have been men-tioned, in this review they are classified into two functions: (i) assubstrates for DNA attachment and (ii) as signal amplifiers. Therole of different nanomaterials in the composition of DNA biosen-sors has recently been intensively reviewed [36]. Therefore, thisreview focuses on the fabrication and validation of portable elec-trochemical DNA biosensors that incorporate NPs as either a signaltransducer or as an electroactive species for the direct detection ofanalytes. Among the different varieties of metal NPs, AuNPs are stillthe most popular NP used in biosensors.

The main reasons of development the method for the detectionof specific target DNA is to fabricate a very sensitive electrochemicalDNA biosensor. The modification of a gold electrode with AuNPs bychemical adsorption of 1,6-hexanedithiol was achieved. The mod-ified electrode was immerged into a mixture of daunomycin andDNA to provide a DNA detection limit at the nanomolar level, mak-ing it one of the most sensitive and convenient approaches [37]. Thepreparation of DNA nanobiosensors by immobilization of a 20-merthiolated probe DNA on a poly(propyleneimine) dendrimer (PPI)doped with AuNPs as the platform on a GCE, resulted in a reversibleelectrochemistry due to the PPI component whilst maintaininga high sensitivity level of 10 pM–5 nM [38]. In a sandwich-typeelectrochemical sensor, AuNPs was used as catalytic labels toachieve ultrasensitive DNA detection via fast catalytic reactions

enabled the detection of as little as 1 fM of target DNA. A novelstrategy using mercaptodiazoaminobenzene-monolayer-modifiedelectrode for assembling AuNPs on the electrode surface was devel-oped since this was believed to improve the electron transfer ability

W.Siangproh

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Table 1Recent reports using AuNPs for the detection of small biomolecules.

Materials Detectiona Analyte Interference Linear range LOD Ref.

AuNPs modified GCE DPV DA and UA AA 0.2–80 �M (DA) 0.12 �M (DA) [15]1.2–100 �M (UA) 0.6 �M (UA)

Au nanorod electrode SWV DA AA 10–100 nM 5.5 nM [16]Au nanocluster modified GCE DPV DA and serotonin AA 7.0–2200 nM (serotonin) 1.0 nM (serotonin) [17]

50 nM–20 �M (DA) 15 nM(DA)Poly(3-

methylthiophene)/AuNPsDPV DA and UA AA 1–35 �M (DA) 240 nM (DA) [18]

1–32 �M (UA) 100 nM (UA)MWCNT-g-

silicaNW/AuNPs/ITODPV DA AA 0.1–30 nM 0.1 nM [19]

AuNPs self-assembly GCE DPV DA AA 10 nM–25 �M 4.0 nM [20]AuNPs self-assembly gold

electrodeDPV DA and AA – 0.3–1.4 mM (AA) 90 �M for both [21]

0.2–1.2 mM (DA)l-Cysteine/AuNPs/GCE CV AA and UA – 2.0–1000 �M (UA) 2.0 �M for both [22]

2.0–800 �M (AA)AuNPs/polyelectrolyte-coated

polystyrene colloidsmodified BDD electrode

CV DA AA 5.0–100 �M 0.8 �M [23]

AuNP/PFG composite GCE Amperometry UA – 1–62 �M 200 nM [24]AuNP/CA/GCE DPV EP AA 0.1–500 �M 40 nM [25]TDPA/CA/AuNPs/Au electrode CV EP AA and UA 0.1–0.65 �M (2D) 0.065 �M (2D) [26]

0.1–0.75 �M (3D) 0.082 �M (3D)MPA/CA/AuNPs/Au electrode CV EP AA and UA 0.1–700 �M (2D) 0.042 �M (2D) [27]

0.1–800 �M (3D) 0.040 �M (3D)AuNPs/PPyox/GEC DPV EP and UA AA 0.3–21 �M (EP) 30 nM (EP) [28]

50 nM–28 �M (UA) 12 nM(UA)AuNPs modified CPE HPLC-amperometry Homocysteine Cysteine, glutathione,

penicillamine andN-acetyl-l-cysteine

0.1–5.0 �M 30 nM [29]

Self-assembled multilayer ofAuNPs

CV Cytochrome c – 1–100 nM 670 pM [30]

AuNPs/CNT/GCE Amperometry Tryptophan – 30 nM–2.5 �M 10 nM [31]FcAI/AuNP/MWCNTs/GCE Amperometry Bilirubin – 1–100 �M 120 nM [32]PASE/Hb/AuNPs/MWCNTs CV Hemoglobin (Hb) – 0.36–17.6 �M 93 nM [33]Aptamer/AuNPs Bio-barcode ASV Adenosine/thrombin – – 6.6 pM (adenosine) [34]

1.0 pM (thrombin)

a Detection methods were; (ASV) anodic stripping voltammetry, (CV) cyclic voltammetry, (DPV) differential pulse voltammetry, and (SWV) square-wave voltammetry.

14 W. Siangproh et al. / Analytica Chim

Table 2Recent uses of AuNPs in voltammetric based sensors for DNA detection.

Materials Linear range LOD Ref.

Daunomycin modified AuNPs – 1.2 nM [37]GCE/PPI/AuNPs 0.01–5 nM – [38]

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DNA-conjugated AuNPs – 1 fM [39]AuNPs-ATP-diazo-ATP/Au 0.3–13.2 nM 91.0 pM [40]AuNPs/MWCNTs/ZnONWs/GCE 0.1 pM–0.1 �M 35 fM [41]AuNPs/PABA-MWCNTs/GCE 1.0 pM–5.0 nM 0.35 pM [42]

f the electroactive species towards the electrode surface, and wasound to provide a very low detection limit of 91.0 pM [40]. More-ver, to enhance not only the sensitivity but also the selectivity,nO nanowires (ZnONWs) [41] were firstly modified onto GCEurface. Next, MWCNTs was used to immobilize on the surface,hen a single-stranded DNA probe with a thiol group at the endHS–ssDNA) was covalently immobilized on the AuNP surface. Thenal sensor had a specific detection limit in the low fM range. Anlternative sensitive (pM range) electrochemical DNA biosensor forhe detection of target DNA was prepared by electropolymerizationf para-aminobenzoic acid (PABA) on the surface of the GCE mod-fied with MWCNTs [42]. AuNPs were subsequently introduced tohe surface of the PABA–MWCNTs composite film, and the probeNA was immobilized on the surface of the AuNPs. Of note is

hat this DNA biosensor had a good stability and reproducibilitys well as a very low detection limit. Table 2 summarizes the ana-ytical figures obtained when using AuNPs in voltammetry for DNAetection.

.3. The use of AuNPs in voltammetry for glucose detection

The ability of AuNPs to provide a stable immobilization ofiomolecules whilst retaining their bioactivity is a major advan-age in the preparation of biosensors. The use of AuNP-basedlectrochemical biosensors has been emphatically reviewed [43],ncluding a critical review of AuNP-based electrochemical glucoseensors. Here, then, the additional published work since that reviews included as shown in Table 3.

Interestingly, it has been described that polymers, such asolyaniline (PANI) can be used with AuNPs and ITO to formuNP–PANI composite films for the fabrication of glucose sensors

44]. It is also reported that an AuNPs–PANI–AgCl/gelatin nanocom-osite pyranose oxidase can be immobilized on the surface of theCE and act as a glucose sensor. However, the linear detection

esponse of this biosensor was narrow and not as sensitive whenompared to other works [45].

Owing to the size effect, the AuNPs in the hybrid material canehave as a good catalyst for both the oxidation and the reduction of2O2, and as such the AuNPs on the surface of AgCl/PANI core–shellanocomposites can be employed with GCE for the highly sen-itive (low pM range) detection of glucose using glucose oxidaseGOx) [46]. Moreover, the resulting AuNP–AgCl/PANI–GOx hybrid

aterial exhibited good electroactivity in a neutral pH environ-ent showing an extremely sensitive response to glucose, with a

etection limit of 4 pM and a linear detection response of up to5 pM.

The other attractive types of glucose sensors are the non-nzymatic glucose electrochemical biosensors, with a low cost,ood stability and reproducibility. The non-enzymatic voltammet-ic glucose sensor can be prepared using AuNPs attached on variousypes of electrodes, including GCE, ionic liquid (IL)-MWCNT and

NT arrays [47–50]. The SEM image of nanocomposites for non-nzymatic glucose determination is shown in Fig. 2.

To get the best benefits from CNTs, a new preparationf AuNP/PSS-functionalized-MWCNT nanocomposites for glucoseiosensing applications have been developed, yielding �M detec-

ica Acta 690 (2011) 10–25

tion levels [51]. A glucose sensor based on the fabrication of GOxonto a crystalline AuNP-modified CNT electrode has also beendeveloped, but again has a low �M detection limit [52]. In addi-tion, a new type of amperometric glucose biosensor has beenconstructed based on the layer-by-layer immobilization of GOx bycross-linking to a chitosan (CS) matrix on a GCE surface [53]. Withincreasing layers of CNT/CS/AuNP, the response current to H2O2was increased.

Several different unique fabrications of glucose sensors usingAuNPs have been developed recently. For example, the incor-poration of genetically engineered periplasmic binding proteins(GGR) that sense glucose on the surface of AuNPs [54], and theamperometric glucose sensor based on the inclusion complex ofmono-6-thio-�-cyclodextrin (CD)/ferrocene (Fc) capped on AuNPs(AuNP/CD–Fc) and GOx [55]. The AuNPs/CD–Fc/GOx glucose sensorshowed a relatively fast response time (5 s), with a good linearityand a detection limit at the micromolar level. The immobilizationof GOx with cross-linking in the matrix of bovine serum albuminon a Pt electrode modified with AuNP-decorated PbNWs was alsodescribed [56]. Moreover, the electrodeposition of the IL biocom-posite film with CS, CS-IL-GOx, onto an AuNPs-modified flat goldelectrode provided a highly sensitive glucose biosensor with a 20-fold lower detection limit than that of the biosensor prepared on aflat gold electrode [57].

2.4. The use of AuNPs in voltammetry for H2O2 detection

H2O2 is a reactive oxygen metabolic by-product that serves as akey regulator for a number of oxidative stress-related states. H2O2is involved in a number of biological events and intracellular path-ways that have been linked to several diseases and, in addition, is anessential mediator in the food, pharmaceutical, clinical, industrialand environmental analyses. Therefore, the reliable, accurate andrapid detection of H2O2 is of considerable practical importance. Todate, there have been many publications covering different meth-ods for the determination of H2O2 concentrations. Metal NPs assensors appear to present some of the most sensitive assays forH2O2 detection, and a recent (2008) review on the use of AuNP-based electrochemical biosensors for detecting H2O2 is available[43]. However, many studies have used different methods for thepreparation of AuNP-based H2O2 sensing, and this is overviewedhere with specific emphasis on the advances in H2O2-detectionmethodologies that use metal NPs as sensors and, in particular,AuNP-based sensors.

ITO is a fascinating material that can be used as a sub-strate due to its biocompatibility. A new method of using AuNPsattached to ITO with myoglobin (Mb) immobilization for the directelectrochemistry and catalysis of H2O2 was reported [58]. The cat-alytic activity of the Mb-immobilized onto the AuNP-modified-ITO(Mb/AuNP/ITO)-electrode showed a good reproducibility and sta-bility in pH 7.0 buffer towards the reduction of H2O2, and a sensitivedetection limit (mid nM) and linear detection range (low-mid �M).Moreover, a new sensor fabricated from a three-dimensionallyordered macroporous AuNP-doped titania (TiO2) film modifiedon the surface of the ITO electrode, and with HRP immobilizedon this film, displayed a rapid electrocatalytic response of lessthan 3 s, high sensitivity, good stability and reproducibility [59]. Inaddition, an interesting disposable pseudo-mediator-less ampero-metric biosensor for the determination of H2O2 levels using HRPcan be prepared using an ITO electrode modified with a thiolfunctional group by (3-mercaptopropyl) trimethoxysilane [60]. The

stable nano-Au-SH monolayer (AuS) was then constructed throughcovalent linkage of the AuNPs and thiol groups on the surface of theITO.

In addition, there are several fabrications of H2O2 sensorsderived from a variety of materials. The nanocomposite of

W. Siangproh et al. / Analytica Chimica Acta 690 (2011) 10–25 15

Table 3Summary of the use of AuNPs in sensors for glucose detection.

Materials Detectiona Interference Linear range LOD Ref.

GOx/AuNPs–PANI/ITO CV – 50–300 mg dL−1 – [44]AuNPs/PANI/AgCl/gelatine Amperometry – 0.05–0.75 mM – [45]GOx/AuNPs–AgCl2/PANI/GCE DPV – 0–35 pM 4 pM [46]AuNW array electrode DPV/amperometry – 2–20 mM (DPV) 30 �M (DPV) [47]

1–10 mM (Amp) 50 �M (Amp)AuNP-CNT array electrode Amperometry – 1–42.5 mM 10 �M [48]AuNPs/MWCNT/IL gel Amperometry UA and AA 5.0–120 �M 2 �M [49]CS-AuNPs/GCE CV – 0.4–10.7 mM 370 �M [50]MWCNTs/PSS/AuNP-IL Amperometry – 0–20 mM 15 �M [51]GOx/AuNPs/MWCNT Hydrodynamic voltammetry – 0–22 mM 30 �M [52]GOx-CNT/CS/AuNPs/GCE Amperometry – 6.0–5000 �M 3.0 �M [53]GCE/AuNPs/GGR/Cys CV – 0–1.0 �M 0.18 �M [54]AuNPs/CD–Fc/GOx Amperometry – 80 �M–11.5 mM 15 �M [55]AuNPs/PbNWs Amperometry – 5–2200 �M 2 �M [56]CS-IL-GOx/AuNPs/Au Amperometry – 3.0 �M–9.0 mM 1.5 �M [57]

a Detection methods were; (CV) cyclic voltammetry, (DPV) and differential pulse voltammetry.

Fig. 2. SEM images of the nanocomposites. (a) GNPs/MWCNTs/IL with different magnifications; (b) MWCNTs/IL; (c) GNPs/IL; (d) GNPs/MWCNTs; (e) GNPs/MWCNTs/IL withmuch more GNPs.From Ref. [49], with permission.

16 W. Siangproh et al. / Analytica Chimica Acta 690 (2011) 10–25

Table 4Summary of H2O2 detection using AuNP based sensors.

Materials Detection Linear range LOD Ref.

Mb/AuNP/ITO Voltammetry 2.5–500 �M 0.48 �M [58]HRP/AuNPs/TiO2/ITO Voltammetry 0.5 �M–1.4 mM 0.2 �M [59]HRP/nano-Au-SH/ITO Voltammetry 5 �M–1.5 mM 1 �M [60]

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HRP/CMCS/AuNPs AmperometryClay/AuNP-CS/GCE VoltammetryHRP/cysteamine-AuNPs AmperometryAuNPs/Au-graphene-HRP-CS/GCE Amperometry

arboxymethyl chitosan (CMCS) and AuNPs was proposed [61].his nanocomposite had the advantage of being hydrophilic even ineutral solutions, stable and inherited the properties of the AuNPsnd CMCS. In addition the biosensor exhibited a fast amperometricesponse.

The nanohybrid material based on clay, CS and AuNPsrepared on a GCE (clay/AuNP–CS), was found to provide aavorable microenvironment for proteins [62]. A one-step pro-edure for the preparation of a HRP–AuNP–silk–fibroin-modifiedCE was developed and the resultant sensors applied totudy the reduction reaction of H2O2 [63]. Another interestingecently developed H2O2 biosensor is a reagent-less amperometriciosensor that is formed by immobilizing the HRP using cross-

inking with cysteamine (CA)-capped AuNPs and then furthermmobilizing this system onto a sodium-alginate-coated Au elec-rode through polyelectrostatic interactions [64]. Lastly, a novel2O2 biosensor based on Au–graphene–HRP–CS biocompositesas successfully prepared [65]. Graphene and HRP were co-

mmobilized into the CS, and the GCE was then modified by theu–graphene–HRP–CS biocomposites followed by electrodeposi-

ion of the AuNPs onto the surface to complete the sensor-electrodeabrication. The characteristics of each fabrication are shownn Table 4.

.5. The use of AuNPs in voltammetry for immunoassays

The main idea of using metal-NP-based electrochemicalmmunoassays is to achieve very high sensitivity in biologi-al assays. The need for ultrasensitive bioanalysis is of majormportance in view of the growing trend of miniaturized method-logy. The achievement of ultrahigh sensitivity requires innovativepproaches that couple different amplification platforms and/ormplification processes. A recent (2007) summary of the use ofetal-NP-based electrochemical detection for immunoassay appli-

ations has already been reported [66], and so in this section these of AuNPs in immunoassays for the electroanalytical verificationf important analytes is reviewed.

Due to the unique properties of AuNPs, antibodies can be self-ssembled onto the surface-confined AuNPs via phenylenediamineusing the amine-Au affinity) with a high loading amount and yettill retain a high immunological activity [67]. Moreover, the com-osites of AuNP/CS and nanogold-MWCNT–Pt–CS composite canlso be employed for the detection of carcinoembryonic antigenCEA) in the low ng mL−1 range [68,69]. In addition, an immunosen-or based on antibody-embedded AuNPs and a SiO2/thionineanocomposition was shown to be almost equally sensitive andighly specific for the detection of CEA [70].

A quasi-reagentless amperometric immunosensor for the deter-ination of prostate-specific antigens (PSAs) in the low ng mL−1

ange was developed [71], where HRP-labeled PSA antibod-

es (anti-PSA) and tetramethyl benzidine were co-adsorbed inuNP–ITO-modified CS membranes.

Likewise, for the convenient and rapid falciparum malaria detec-ion, a disposable amperometric immunosensor was developedor the detection of Plasmodium falciparum histidine-rich protein

5.0 �M–1.4 mM 0.401 �M [61]39 �M–3 mM 7.5 �M [62]20 �M–13.7 mM 3 �M [64]5 �M–5.13 mM 1.7 �M [65]

2 (PfHRP-2) in human sera at a low pg mL−1 level using screen-printed electrodes (SPEs) modified with MWCNTs and AuNPs [72].

Using the concept of hybrid materials [73], AuNP/C electrodescan be conjugated with HRP-labeled antibody (HRP–Ab) to fabri-cate HRP–Ab–AuNP/C bioconjugates, which can then be used as alabel for the sensitive detection of the target antigen. This approachprovided a very low detection limit of 5.6 pg mL−1 for the detectionof specific proteins [73]. In addition, AuNP/CNT-hybrid-modifiedbiosensors have been used for the sensitive detection of humanIgG at levels as low as 40 pg mL−1 [74].

Another interesting fabrication method is the electropolymer-izing of a conducting polymer, poly-terthiophene carboxylic acid(poly-TTCA) in this case, onto a AuNP/GCE, and covalently bond-ing a dendrimer (Den) to the poly-TTCA [75]. These amperometricimmunosensors have been used to diagnose lung cancer throughthe detection of Annexin II and MUC5AC antigens at the pg mL−1

level.Using a sandwich-type immunoassay format, the sensitivity of

the electrochemical methods can be enhanced by AuNP-labeleddetection [76]. In this case, using the hepatitis B surface anti-body (HBsAb) immobilized on a AuNP/thionine/DNA-modified goldelectrode, a sandwich-type immunoassay was then used for thedetection of HBsAg at low pg mL−1 levels using AuNP-modifiedHRP–HBsAb conjugates as secondary antibodies. Other similarwork for the detection of alpha-fetoprotein (AFP), but based ona CPE constructed of room-temperature IL-AuNPs, allowed detec-tion levels as low as 250 pg mL−1 [77]. By sandwiching the antigenbetween the anti-AFP Ab on the AuNP–CPE and the secondary HRP-polyclonal anti-human-AFP Ab, the immunoassay was established.Fig. 3 demonstrates the schematic of the stepwise immunosensorsfabrication process.

Besides these sandwich-type immunoassays, a competitiveimmunoassay format can be used for the detection of the same ana-lyte. Here, AFP detection with a CNT–AuNP-doped CS film attaineda detection limit from 0.6 ng mL−1 [78]. Other analytes, such asbiotin, can also be detected by competitive-type immunoanalyt-ical platform at low pg mL−1 levels, providing a cost-effective,easy-to-use method [79]. The competition occurs between theanalyte biotin and potassium ferrocyanide-encapsulated, biotin-tagged liposomes immobilized on the nanoAu/SPE surface.

Another new approach towards the development ofadvanced immunosensors based on chemically functionalizedcore–shell–shell nanocomposite particles was found to not onlyhave the properties of magnetic nanoparticles but also providedgood biocompatibility for the immobilization of biomolecules[80]. The core–shell–shell nanostructure presents good magneticproperties that facilitate and modulate its integration into aCPE. Moreover, the magnetic core/shell particles coated withself-assembled AuNP multilayers have been used to detect CEAantigens with a very low limit detection limit (1 pg mL−1) [81].

Recently, a new electrochemical immunoassay was formulatedby direct linking of the antibody to the AuNPs using a spacerarm [82], which provides a novel means for antibody immobiliza-tion and the study of antibody-antigen interactions with a target(H2O2) sensitivity of ∼2 ng mL−1. Ding et al. [83] reported on the

W. Siangproh et al. / Analytica Chimica Acta 690 (2011) 10–25 17

epwisF

cgbs

itictCb

digdA

dpepFtl

oAa(

uAka[m

2

itdad

Fig. 3. Schematic illustration of the strom Ref. [77], with permission.

ombination of electrochemical immunoassay using a nanoporousold (NPG) rod electrode with HRP-labeled secondary Ab-AuNPsioconjugates for highly sensitive detection of proteins in serum,uch as HBsAg at ∼2 pg mL−1.

The label-free electrochemical immunoassay based on AuNPss another approach that is very useful for bioanalytical applica-ions. Tang et al. [84] described one such label-free electrochemicalmmunoassay for the detection of CEA based on AuNPs and non-onductive polymer films. Anti-CEA Abs were covalently attachedo a glutathione monolayer-modified AuNP and the resulting anti-EA–Ab–AuNP bioconjugates were immobilized on a Au electrodey electro-copolymerization with o-aminophenol.

A new Ab immobilization strategy based on the electro-eposition of AuNPs and Prussian Blue as a label-free amperometric

mmunosensor for the determination of the hepatitis B surface anti-en (HBsAg) revealed a sensitive (400 pg mL−1) and broader linearetection range (2–300 ng mL−1) [85], than that seen with NPG-uNPs [83].

Additionally, a label-free electrochemical immunosensor for theetection of specific DNA using the Fc-containing cationic polythio-hene and peptide nucleic acid (PNA) probes on AuNPs modifiedlectrodes increased the immobilized amount of ss-PNA capturerobe and lead to an increase of the electrical signal [86]. Likewise,ang et al. [87] developed a label-free sensor for adenosine detec-ion with a high sensitivity, desirable selectivity and a 30-fold wideinear range.

More recently, a label-free amperometric immunosensor basedn the immobilization of the Ab on a positively chargeduNP/l-cysteine-modified gold electrode was developed for themperometry based determination of IgG serum levels in humans0.5–25 ng mL−1) in the absence of a label [88].

Alternatively, novel label-free immunosensors can be preparedsing functionalized AuNPs by covalently capping the surface ofuNPs with 1,1′-bis-(2-mercapto)-4,4′-bipyridinium dibromide, aind of sulhydryl viologen (SV), for the detection of AFP and attainedsimilar sensitivity (230 pg mL−1) and broad linear detection range

89]. The analytical performance of these immunosensors is sum-arized as shown in Table 5.

.6. The use of quantum dots in voltammetry

A quantum dot is a semiconductor whose excitons are confined

n all three spatial dimensions. As a result, they have proper-ies that are between those of bulk semiconductors and those ofiscrete molecules. Quantum dots have been used in many bio-nalytical applications, such as immunoassays and DNA detection,ue to their large surface area and good stability. Even though the

e immunosensor fabrication process.

modification of biological molecules onto the quantum dots is notalways easy, the anodic stripping voltammetry of quantum dotsprovides a high sensitivity and selectivity. The most importantadvantage of quantum dots over AuNPs is the simplicity of per-forming multiple analyte detections using the differences in theanodic stripping potential of the different analytes with differentmetal quantum dots, such as CdS, PbS and ZnS. The most com-monly used quantum dot system is the modification of quantumdot outer shell with a carboxyl or amine group before bindingwith protein or DNA. The application of oligonucleotide-conjugatedCdSNPs as probes for the electrochemical detection of the nopalinesynthase (NOS) terminator gene sequences resulted in a relativelylow detection limit and a three-log order linear detection range[90]. Here, mercaptoacetic acid-modified CdSNPs were first syn-thesized, and then the carboxyl group on the surface of the CdSNPswere covalently bound with the NH2-modified NOS oligonucleotideprobe sequences. Nitric acid was then used to oxidize the CdSNPsanchored on the hybrids and was further detected by a differentialpulse anodic stripping voltammetric method.

Another example of the use of outer shell modification iscadmium selenide (CdSe) quantum dots modified with ethylene-diamine and labeled onto the probe DNA [91]. The CNTP electrodewas modified with AuNPs, and the target DNA was immobilizedon the AuNP membrane electrode and hybridized with the probeDNA labeled with CdSe. Next, CdSe was dissolved in HNO3 and thetarget DNA was detected by the differential pulse anodic strippingvoltammetry of Cd, where a 650 fM detection limit and a four logorder linear detection range (pM–nM) was obtained.

However, to obtain a very sensitive method, quantum dots canbe modified with a hairpin probe [92] or poly(styrene-co-acrylicacid) (pSTcAA) microbeads [93]. These modifications provide anultra-low detection limit, down to the fM level of the target DNA(Table 6).

2.7. The use of other metal NPs in voltammetry

Owing to the unique properties of metal NPs, various types ofmetal NPs have been synthesized for use as sensor components toenhance the detection sensitivity of the target analyte(s). In thissection, we review the advances in the use of other metal NPs involtammetric based bio-analysis.

Platinum NPs (PtNPs) are the principal alternative metal NPs

for anodic current measurement because of their strong catalyticoxidation. Novel electrodes modified with an electrodeposited filmcontaining DNA and Pt nanocomposites was developed for selec-tive anodic voltammetric assays that can resolve the overlappingvoltammetric responses of DA, UA and AA into three well-defined

18 W. Siangproh et al. / Analytica Chimica Acta 690 (2011) 10–25

Table 5Summarized data of AuNPs used as detectors in voltammetric based immunoassays.

Materials Detectiona Analyte Linear range LOD Ref.

Fc/PPD/AuNPs Amperometry IgG 25–1000 pg mL−1 10 pg mL−1 [67]AuNPs-CS/GCE Amperometry CEA 0.2–120.0 ng mL−1 0.06 ng mL−1 [68]AuNPs/MWNT-Pt-CS Amperometry CEA 0.5–10 ng mL−1 0.2 ng mL−1 [69]

10–120 ng mL−1

AuNPs/SiO2/Thionine Amperometry CEA 1.00–100.00 ng mL−1 0.34 ng mL−1 [70]AuNPs-CS/ITO Amperometry PSA 5.0–30 ng mL−1 1.0 ng mL−1 [71]AuNPs/MWCNT/SPEs Amperometry PfHRP-2 – 8 ng mL−1 [72]HRP-Ab-AuNP/C Amperometry Protein 0.01–250 ng mL−1 5.6 pg mL−1 [73]AuNPs/CNTs/HPR/GCE Amperometry Human IgG 0.125–80 ng mL−1 40 pg mL−1 [74]Den-pTTCA/AuNP/GCE Amperometry Annexin II and MUC5AC – 51 pg mL−1 [75]HBsAb/AuNPs/thionine/DNA/Au Amperometry Hepatitis B 0.5–650 ng mL−1 0.1 ng mL−1 [76]IL/AuNPs/CPE DPV AFP 0.50–80 ng mL−1 0.25 ng mL−1 [77]AuNPs/CNT/CS Amperometry AFP 1–55 ng mL−1 0.6 ng mL−1 [78]PAH/AuNPs/SPE SWV Biotin 1–10 pM 9.1 pg [79]Anti-AFP/AuNP/NiFe2O4/APTES Amperometry AFP 0.9–110 ng mL−1 0.5 ng mL−1 [80]AuNPs/Fe3O4 Amperometry CEA 0.005–50 ng mL−1 1 pg mL−1 [81]AuNPs/Ab1/Au CV H2O2 – 2 ng mL−1 [82]AuNPs/NPG rods Voltammetry HBsAg 0.01–1.0 ng mL−1 2.3 pg mL−1 [83]AuNPs/Prussian Blue/GCE Amperometry HBsAg 2–300 ng mL−1 0.42 ng mL−1 [85]

amme

pr

csgateom

bNoliIt(edo[tPaoos

pa

TS

Anti-hIgG/AuNP+s/l-cysteine/Au Amperometry Human-IgGSV-AuNPs Amperometry AFP

a Detection methods were; (CV) cyclic voltammetry, (DPV) differential pulse volt

eaks [94,95], yet demonstrated a good sensitivity (typically nMange), selectivity and stability.

Also using PtNPs, the effective electrocatalytic oxidation of glu-ose with a PtNP-based carbon electrode was developed, which,howed a significant positive catalytic effect for the oxidation oflucose compared to that with the AuNP based system [96]. Inddition, nanocomposites of ethylene glycol protected PtNPs inhe presence of activated carbon were developed for the non-nzymatic detection and quantification (2–20 mM detection limits)f glucose over its physiological range using hydrodynamic voltam-etry [97].Palladium NPs (PdNPs) have been used for amperometric

iosensors. Polymer-stabilized PdNPs on a carbon support withafion resulted in a glucose biosensor with an improved stabilityf both the ink and biosensor electrode and a reduced interferenceevel [98]. An alternative way to prepare PdNPs modified electrodess by electrochemical deposition [99]. The nano Pd film modifiedTO-GCE exhibited electro-oxidation signals that were effective forhe discriminative detection of catecholamines, EP, norepinephrineNEP) and DA in the low �M range. Moreover, PdNPs modifiedlectrodes, prepared by distributing PtNPs or PdNPs into a con-uctive polymer matrix of poly(3-methylthiophene) (PMT) [100],r by electrospinning and subsequent thermal treatment of a CPE101], have been used as sensors for the simultaneous determina-ion of DA, UA and AA with high nM to low �M detection limits. ThedNP/CNF modified CPE displayed excellent electrochemical cat-lytic activities towards DA, UA and AA with significantly decreasedxidation overpotentials of all three analytes compared with thosebtained by the bare CPE. This nanosensor exhibited a remarkable

ensitivity and a wide linear detection range for all three analytes.

Silver NPs (AgNPs) have recently become one of the mostopular materials to fabricate sensors for variety of compoundss they exhibit high extinction coefficients and are capable of

able 6ummarization of the use of quantum dots in voltammetry based DNA detection.

Materials Detectiona Analyt

MMA/CdSNPs DPASV NOSAuNPs/CdSe/CNTP DPASV DNACdTe-modified hairpin probe/Au Voltammetry DNACdTe-tagged pSTcAA microbeads SV DNA

a Detection methods were; (DPASV) differential pulse aniodic stripping voltammetry, (

0.5–25 ng mL−1 – [88]1.25–200 ng mL−1 0.23 ng mL−1 [89]

try and (SWV) square wave voltammetry.

facilitating the electron transfer reactivity of some biologicalmolecules. Thus, when AgNPs were modified onto an electrode sur-face, the electrochemical response of guanosine and guanine canclearly revealed the activity of the purine nucleoside phosphory-lase (PNP) enzyme from a detection limit of 0.1 U mL−1 [102]. Theobtained linear detection range (4–20 U mL−1) and limit are goodenough for clinical applications. Additionally, an AgNP-modifiedcomposite electrode has been developed for the sensitive electro-analysis of glucose [103].

Based on a similar concept for composite electrodes,MWCNT–AgNP composites were developed for the detectionof AFP using MnO2–CS–GCE [104] and H2O2 [105], since thehigh surface area of MWCNTs provides a large loading capacityfor nanoparticles and revealed a much improved sensitivity andcatalytic activity.

Moreover, Hb co-immobilized with Ag-AgONPs on a bare sil-ver electrode revealed a nearly four-log order linear reductionrange for detection of H2O2 at the co-immobilized electrodes,with a detection limit of ∼2 �M [106]. Conducting polymers havebeen established as suitable host matrices for dispersing metal-lic particles. A new material that can effectively overcome thekinetic barriers for the oxidation of NADH was created by usinga Meldola-Blue poly(3,4-ethylenedioxythiophene) (PEDOT) immo-bilized AgNP-conducting polymer electrode [107]. The detectionlimit was found to be 0.1 �M. The benefit of this modified sensor isdiminished level of interference.

Copper NPs (CuNPs) have been used as a detector in a high-performance amperometric glucose sensor [108], which exhibitedan excellent sensitivity (20 �M detection limit), a fast response time

(<4 s), a wide linear detection range (50 �M–12 mM) and perfectselectivity. Moreover, CuNPs have also been incorporated into aPPoyx modified GCE as a sensor for the highly sensitive simulta-neous determination of DA and UA levels at a high pM level and

e Linear range LOD Ref.

8.0 pM–4.0 nM 2.75 pM [90]5.0 pM–500 nM 0.65 pM [91]– 4.7 fM [92]– 0.52 fM [93]

SV) stripping voltammetry.

W. Siangproh et al. / Analytica Chimica Acta 690 (2011) 10–25 19

Fig. 4. AFM images of the (A) bare GCE, (B) PPy/GCE, and (C) nano-Cu/PPy/GCEsF

wAIt

bshpaa

c[sesb

Fig. 5. Amperometric responses of the Mb/Fe3O4@Au/MGCE at −0.35 V to suc-cessive addition of H2O2 in a stirred 0.1 M PBS (pH 6.98). Inset: (A) cyclicvoltammograms of the Mb/Fe O @Au/MGCE in 0.1 M pH 6.98 PBS containing 0 (a),

sulphonated polyaniline (SPAN) nanofiber [137] and ZnONW [138]were found to provide enhanced Ab immobilization and a higher

urfaces as three-dimensional views.rom Ref. [109], with permission.

ith a four-log order linear detection range [109]. Fig. 4 shows theFM image and film thickness of the bare and modified electrode.

t can be seen that the CuNPs were homogeneously distributedhroughout the film.

Rhodium NPs (RhNPs) are another type of metallic NPs that haveeen broadly used for catalysis and sensing of biological molecules,uch as cytochrome c and H2O2. Typically, the synthesis of RhNPsas depended on the type of stabilizer used to help the NP dis-ersion, but a cobalt aminophthalocyanine macrocyclic complexnd a N,N-bis-succinamide-based dendrimer have been reporteds suitable stabilizers for the preparation of RhNPs [110,111].

Other metal NPs, including selenium [112], nickel [113–115],erium oxide [116,117] copper oxide, [118,119], ZnO [120], TiO2121,122] and Fe3O4 [123–126], also have good potential in biosen-or development in terms of their biocompatibility and high

lectron mobility. In addition, these sensors exhibited a goodensitivity, selectivity, stability and long-term maintenance ofioactivity.

3 4

15 (b), 50 (c), and 100 �M (d) H2O2 at 100 mV s−1. (B) Plot of chronoamperometriccurrent vs. H2O2 concentration.From Ref. [132], with permission.

Currently, bi-metallic NPs are attracting increasing interestbecause they feature interesting catalytic behaviours with respectto that seen with monometallic systems. Compared with sin-gle metal NPs, bimetal NPs present distinct characteristics, inwhich one metal takes on a long-term stability and biocom-patibility, and the other shows the specific optical or electricalactivity to be monitored. Most reports regarding bimetallic NPsare related to the combination of Au with another metal, suchas PbS [127], Cu [128], Pt [129,130], ZnO [131] and Fe3O4 [132].These bi-metallic electrodes typically provide a rapid response,good stability, electrocatalytic and reproducibility for the selectedtarget detection. Fig. 5 illustrates the bioelectrocatalytic activity ofMb/Fe3O4/AuNP/magnetic GCE toward the reduction of H2O2. Thereduction peak current increased as a function of H2O2 concentra-tion, displaying obvious electrocatalytic behaviour of Mb.

The simplified methodology mentioned for the preparationof NP sensors showed the potential application for fabricatingnovel biosensors and bioelectronic devices for the (bio)analyticalresearch field, and are summarized in Table 7.

For other electrochemical techniques, potentiometric detectionis based on the measurement of electrical potentials without draw-ing appreciable current. The electrical potentials are related to thequantity of the analyte of interest, which is generally its concentra-tion in the test solution. Potentiometric immunosensors generallyinvolve the immobilization of immunoactive antibodies or simi-lar probes onto the electrochemical transducer. With the adventof miniaturized electrode technology, the deposition of differentNP-based biocompatible films on the electrode surface is a greatchallenge.

The immobilization of FeNPs onto the electrode surface can beused expediently, renewed easily and forms a relatively low-costimmunosensor. Based upon this principal, renewable potentiomet-ric immunosensors were developed that showed a better stabilityand a higher sensitivity for immunoglobulin G (IgG) detection ata 23 pg mL−1 limit using Fe3O4NPs [133], alpha-fetoprotein (AFP)detection at a 300 pg mL−1 limit using CoFe2O4 [134] and CEAat 0.9 ng mL−1 using Fe3O4 nanorods [135]. Furthermore, PPy-AuNPs [136], Cysteamine-capped gold nanoparticle (CA-G(NP)) and

sensitivity for immunosensors. The use of AgNPs as redox mark-ers has also been used to detect glucose (10 �M detection limit) bya potentiometric approach based on polymeric membrane Ag-ISE

20 W. Siangproh et al. / Analytica Chimica Acta 690 (2011) 10–25

Table 7Summary of recent reports on biomolecule detection using various metal NPs.

Materials Detectiona Analyte Interference Linear range LOD Ref.

Pt-Fe(III)/GCE DPV UA AA 3.8–160 �M 1.8 �M [94]DNA/Pt nanocluster modified electrode DPV DA and UA AA 0.11–38 �M (DA) 36 nM (DA) [95]

0.3–57 �M (UA) 100 nM (UA)Pt-C, Pt-MWCNT and Pt-CNF Hydrodynamic

voltammetryGlucose – 2–20 mM – [97]

PdNPs-ITO-GCE electrode ADPV Catechola-mines, EP,NEP and DA

AA 34–279 �M (EP) – [99]

9–126 �M (NEP)8–88 �M (DA)

PtNPs or PdNPs/(PMT) Voltammetry DA and AA UA and Glucose 0.05–1.0 �M (DA) – [100]Pd/CNFs modified CPE DPV DA, UA and AA – 0.5–160 �M (DA) 0.2 �M (DA) [101]

2–200 �M (UA) 0.7 �M (UA)0.05–4 mM (AA) 15 �M (AA)

AgNPs modified electrode – PNP – 4–20 U mL−1 0.1 U mL−1 [102]CS-MnO2/MWCNT-AgNP/GCE CV AFP – 0.25–250 ng mL−1 0.08 ng mL−1 [104]MWCNT/AgNP/Au Voltammetry H2O2 AA and UA 0.05–17 mM 500 nM [105]Hb/Ag-AgONPs/Ag Voltammetry H2O2 – 6.0 �M–50 mM 2.0 �M [106]PEDOT/AgNPs Amperometry NADH – 10–560 �M 0.1 �M [107]GOx/CuNPs/CS/CNT/GCE Amperometry Glucose – 0.05–12 mM 20 �M [108]CuNPs/PPoyx/GCE DPV UA and DA – 1.0 nM–10 �М (UA) 0.8 nM (UA) [109]

1.0–100 nM (DA) 0.85 nM (DA)RhNP/GCE DPV Cytochrome c – 100 nM–3 �M – [110]RhNP-DENs/GCE Amperometry H2O2 – 8–30 �M 5 �M [111]SeNPs Amperometry H2O2 – 8 nM–80 �M 80 nM [112]NiNP-CNT-CS Amperometry Glucose – 0.05–10 mM 10 �M [113]HRP/NiONPs/GCE CV H2O2 – 1.0–10 mM 123 �M [114]C-coated magnetic NiNP-GCE CV/DPV NEP – 0.2–80 �M 60 nM [115]CeO2NP-modified electrode DPV Rutin – 0.5–500 �M 0.2 �M [116]CeO2NP/GCE Voltammetry UA – 0.2–500 �M 0.1 �M [117]CuONP-modified electrode CV/DPV Rutin – 0.1–500 �M – [118]CuONPs/MWCNTs Amperometry Glucose – up to 3 mM 800 nM [119]Enzyme/ZnONPs/CNTs DPV UA – 5.0 �M–1 mM 2.0 �M [120]Anti-AFP/NGP/PV-TiO2NP Voltammetry AFP – 1.25–200 ng mL−1 0.6 ng mL−1 [121]TiO2NP/CPE DPV Buzepide – 50 nM–50 �M 8.2 nM [122]Fe3O4NPs/GOx/Pt/CS Volt Glucose – 6.0–2200 �M 6.0 �M [123]Fe2O3NPs/C-IL/Mb-modified electrode CV TCA/NaNO2 – 0.6–1.2 mM (TCA) 0.4 mM (TCA) [124]

4.0–100 mM (NaNO2) 1.3 mM (NaNO2)SiO2/Fe3O4NP/MWCNT CV/amperometry Glucose – 1 �M–30 mM 800 nM [125]NiFe2O4NPs/CS CV Glucose – 0.1–20 mM – [126]PbSNPs/Au DPASV DNA – 0.9–70 fM 0.26 fM [127]Core–shell Cu/AuNPs ASV E. coli – 50–500 kCFU mL−1 30 CFU mL−1 [128]Pt-Au nanoporous film CV/amperometry E. coli – 20–106 CFU mL−1 10 CFU mL−1 [129]AuNP/PtNP-TiO2-nanotubular electrode Voltammetry Glucose – 0–1.8 mM 0.1 mM [130]ZnO-AuNPs-Nafion-HRP modified GCE Voltammetry Glucose – 15–1100 �M 9.0 �M [131]

nionics ltamm

feiac

letFttocal[us

nH

Mb/Fe3O4/AuNP/magnetic GCE Voltammetry H2O2

a Detection methods were; (ADPV) anionic differential pulse voltammetry, (ASV) atripping voltammetry, (DPV) differential pulse voltammetry, and (SV) stripping vo

abricated from benzothiazole calix[4]arene [139]. The H2O2 gen-rated from the GOx was able to oxidize the AgNPs to free Ag+ ionsn proportion to the concentration of glucose, and so Ag+ ion levels,s directly monitored using the Ag-ISE, were related to the glucoseoncentration.

Capacitive immunoassays are based on the electrical double-ayer theory. An electrode that is coated with a biorecognitionlement has a stable capacitance signal that will decrease whenhe analyte binds to the biorecognition element on the electrode.or a biosensor based on a capacitive transducer, the immobiliza-ion of the recognition element is of vital importance to the abilityo detect the binding event. A label-free immunosensor, basedn a modified electrode incorporated with AgNPs to enhance theapacitive response to microcystin-LR (MCLR), was found to have1.7-fold higher sensitivity (10 pg–1 �g L−1) and a lower detection

imit (7 pg L−1) compared to the modified electrode without AgNPs140]. Another example of a novel capacitive immunosensor is the

se of a AuNPs monolayer on a GCE for the detection of Salmonellapp., with a detection limit of 100 CFU mL−1 [141].

Electrochemical impedance spectroscopy (EIS) is another tech-ique that has been used to study electrochemical systems.owever, impedance methods have only recently been applied

– 1.28–283 �M – [132]

stripping voltammetry, (CV) cyclic voltammetry, (DPASV) differential pulse anionicetry.

in the field of biosensors. AuNPs and AuNPs/CNT-modified elec-trodes are widely used materials in impedance sensors and involvethe incorporation into an ensembled substrate, which a pro-tein, oligonucleotide, or other probe molecule is incorporatedto amplify the impedance signals [142,143]. The NPs can beformed as NP-biomolecule conjugates either in the solution orafter being modified on the electrode surface. The constructionof three-dimensional networks with AuNPs dispersed throughoutthe sensing interface is an important criterion for using AuNPs toenhance the impedance signal in biosensors. In addition, the useof CNT incorporated within the sensing interface leads to a muchgreater signal enhancement due to their high conductivity andactive surface area.

Conductometric biosensors are a very promising class of analyt-ical devices characterized by having a high sensitivity. They permitthe detection of conductance changes that result from the reac-tions catalyzed by an immobilized enzyme on the sensor surface. A

large number of enzymes are known to produce ionic products thatincrease the conductivity, but there are also enzymes, for exampleGOx, whose reaction products induce a net decrease in the conduc-tivity. Using this concept, a conductometric immunosensor basedon NPs for the detection of Escherichia coli was developed with a

W. Siangproh et al. / Analytica Chimica Acta 690 (2011) 10–25 21

Table 8Summarized information of using metal NPs for bioanalytical applications by other electrochemical techniques.

Materials Detectiona Analyte Linear range LOD Ref.

Anti-IgG/cysteine/Fe3O4NPs Potentiometry IgG 0.1–1.2 ng mL−1 23 pg mL−1 [133]Anti-AFP/AuNPs/CoFe2O4-MPS Potentiometry AFP 0.8–120 ng mL−1 0.3 ng mL−1 [134]Anti-CEA/Fe3O4 nanorods/CPE Potentiometry CEA 1.5–80 ng mL−1 0.9 ng mL−1 [135]PPy/AuNPs/Au Potentiometry Hb and Hb-A1c 60–180 �g mL−1 (Hb) and

4–18 �g mL−1 (Hb-A1c)– [136]

CA-G(NP)/SPAN (n) on Au/MPA electrode Potentiometry/Chronopotentiometry/EIS

DNA Up to 213 fM – [137]

ZnONW/Ag Potentiometry Glucose 0.5–1000 �M – [138]AgNPs-ISE Potentiometry Glucose 0.1–3 mM 10 �M [139]AgNPs/anti-MCLR Capacitive Method MCLR 10 pg L−1–1 �g L−1 7.0 pg L−1 [140]AuNPs/GCE Capacitive Method Salmonella spp. 0.1–100 kCFU mL−1 100 CFU mL−1 [141]Magnetite NPs, Conductometry E. coli – 1 CFU mL−1 [144]AuNPs QCM C-reactive protein Up to 100 �M 87 fM [145]

HumHum

mical

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CdSe NPs/CNT-CS ECLGoat-anti-human IgG/AuNP Sandwich-ECL

a Detection methods were; (ECL) electrochemical luminescence, (EIS) electroche

ood response in terms of the detection of the amount of antigenE. coli) added (1 CFU mL−1 detection limit) and a high selectivity144]. Quartz crystal microbalance immunosensors have also beensed as detectors in biological systems using AuNP-based signalmplification [145]. The signal augmentation after AuNPs bindingmounted to 53.4% compared with the absence of AuNPs, and thisesulted in a significant improvement in the sensitivity of the cur-ent immunosensor, allowing detection of the C-reactive proteinrom 87 fM.

Another immunosensor that involves the use of NPs is the elec-rochemiluminescence (ECL) immunosensor. There are many kindsf NPs, such as the bimetallic Pt-AuNPs [146], CdSe nanocompos-tes [147] and AuNPs [148], that have been immobilized onto thelectrode surface for use in ECL based detection. The enhancementf ECL signals is due to the increase in the specific surface area ashe size of the NPs diminishes, resulting in a higher surface activ-ty. Thus, the smallest NPs typically have the highest efficiency.able 8 summarizes the reported information and analytical per-ormances obtained from using metal NPs as one part of sensor toetect biomolecules using various electrochemical techniques.

. Metal-NP-based electrochemical detection ininiaturized systems

Recently, miniaturized systems and analyses have become pow-rful tools in chemical and biological systems due to their higherformance, design flexibility, reagent and sample economy,igh throughput, miniaturization and automation [149–151]. Thus,icrofluidic devices can dramatically alter the speed and scale of

nalyses. In biosensing systems in particular, the use of microflu-dic chip-based sensors has outstanding advantages, including aast response and analysis of highly selective biochemical reactions152]. The integration of metal-NP-based electrochemical detec-ion with microfluidic chips offers a significant advantage becauset combines sensitive electrochemical detection with a compact

icrofluidic platform, thus capitalizing on the benefits of both tech-ologies. In the following section, we review the use of metal NPs asetectors in miniaturization systems using voltammetric and otherlectrochemical techniques.

In voltammetry, a potential sweep is applied to a working elec-rode, and the current resulting from the reduction or oxidationf the redox-active species on the electrode surface is measured.

oltammetry is advantageous because it is sensitive, versatile,imple and robust. Compared to potentiometry, voltammetry isess strongly influenced by electrical disturbances and thus has

more favorable signal-to-noise ratio. In addition, voltamme-ry offers a number of different analytical techniques, including

an IgG 0.02–200 ng mL−1 1 pg mL−1 [147]an IgG 5.0–100 ng mL−1 1.68 ng mL−1 [148]

impedance spectroscopy, (QCM) quartz crystal microbalance.

linear, cyclic, stripping and amperometric methods, which eachyield different types of information. For example, cyclic voltam-metry also provides information about the diffusion coefficientsof the charged species. In addition, the use of different typesof metal in the working electrode or different stripping tech-niques also provides more information. The combination of thedifferent working electrode compositions and the analytical andstripping techniques allows access to many types of sampleinformation.

Based on the current literature, there are at present two tar-gets for using metal NPs in miniaturized system. The first is theutility of metal NPs in the separation sciences [153–156], whichare the most common applications at present. This is due to thelarge surface-to-volume ratio of the NPs, an important parameter inchromatography, which allows the NPs to achieve more favorableperformances. Fairly recently (2007), Nilsson et al. [155] reviewedthe use of NPs in capillary and microchip capillary electrophore-sis; therefore, this review covers the use of NPs as the stationaryor pseudostationary phase in capillary and microchip electrochro-matography. To focus on electrochemical detection, the use ofgold NPs (AuNPs) modified on a microchannel to improve selectiv-ity in miniaturized system before detection by amperometry wasalso described [153]. Using the same idea, the development of amicrochip-based aptasensor for the detection of human thrombinwith a detection limit of as low as 1 pM was recently reported [154].Fig. 6 demonstrates schematic representation of the aptamer-basedassay in the microfluidic microchannel as well as the placement ofelectrode on the microchip.

The second target is the use of metal NPs as electrochemicalsensors to enhance the signal in miniaturized system. Surprisingly,metal-NP-based electrodes have so far rarely been employed assensors for microfluidic systems until recently, so there are onlya few current publications available on this subject. In 2004, theuse of copper NPs (CuNPs) in a modified carbon-nanotube paste(CNTP) as the detection electrode in microchip electrophoresis forthe electrocatalytic oxidation of carbohydrates and amino acidswas reported [157]. Using the CuNPs, the sensitivity enhancementled to a very low detection limit of 1 nM, whilst the NP compositeelectrodes demonstrated excellent resistance to electrode fouling,unlike bulk copper electrodes.

Gold NPs were also used as an electrochemical label for multian-alyte determination by a disposable chip [158]. The immunosensors

array as the disposable chip was firstly prepared by immobiliz-ing capture antibodies on different screen printed carbon workingelectrodes by passive adsorption as shown in Fig. 7. Using humanIgG and goat IgG as model targets, under optimal conditionsthis method achieved linear ranges from 5.0 to 500 and 5.0 to

22 W. Siangproh et al. / Analytica Chimica Acta 690 (2011) 10–25

idic mF

4r

woemweaoTrbals

F

Fig. 6. (A) Schematic representation of the aptamer-based assay in the microflurom Ref. [154], with permission.

00 ng mL−1 with low limits of detection of 1.1 and 1.6 ng mL−1,espectively.

Another example of using NPs as detectors comes from theork of Shiddiky’s group [159], who described the development

f a simple and sensitive on-chip preconcentration, separation andlectrochemical detection of trace DNA. In this work, a poly(methylethacrylate) (PMMA) microchip system with an embedded AuNPorking electrode was designed. AuNPs were not only utilized to

nhance the performance of preconcentration and separation byddition into the buffer but were also electrochemically modifiednto a working electrode to improve the sensitivity of method.he modified microelectrode demonstrated a high sensitivity andeproducibility compared to that obtained with the corresponding

are electrode. The combined methodology resulted in a remark-ble increase in the sensitivity by ∼25,000-fold, giving a detectionimit as low as at the attomole level of sensitivity. Therefore, theensitivity, efficiency, selectivity, reproducibility, miniaturization

Fig. 7. Schematic representation of the preparation of immunosensors array anrom Ref. [158], with permission.

icrochannel, and (B) sketch of the microfluidic chip showing electrode location.

and decreased reagent/sample consumption suggests the possibil-ity of performing field analysis and diagnosis of diseases in an earlystate.

Subsequentially, the development of a simple device for rapidbiosensing (in this case, for hydrogen peroxide (H2O2) as themodel analyte) utilizing a single microfluidic channel made froma polymer chip and incorporating an electrochemical system wasproposed [160]. The working electrode was a gold wire coatedwith 5-nm glutathione-decorated AuNPs. In contrast to the ear-lier (above-mentioned) work, the main idea in this study for usingAuNPs was to improve the sensitivity of the detection unit. H2O2was used as the model analyte because it is a frequent productin many biosensors, but the principal could be adapted to other

analytes. Interestingly, this sensor provided a low (5 nM) detec-tion limit in unmediated biocatalysis with a sensitivity level of1.46 nA nM−1 cm−2. The detection limit of the microfluidic sensorwas better than that found previously work using a rotating-disc

d analytical procedure for the simultaneous detection of HIgG and GIgG.

W. Siangproh et al. / Analytica Chim

Fig. 8. Integration of both analysis and calibration for some pharmaceuticals: designosaF

poc

mctNd

uotcrrTbpcssCwisiqcdCg

f strategy with photograph of MWCNTs as detectors (610,000) (A). Analysis ofupplements: Hidrosil (B), Aspol (C), and Becozyme (D). Peaks: (1) pyridoxine, (2)scorbic acid, and (3) folic acid.rom Ref. [162], with permission.

oly(diallyldimethylammonium chloride)/AuNPs/horseradish per-xidase (PDDA/AuNPs/HRP) biosensors in a bulk electrochemicalell.

Recently, the fundamentals, designs and applications of nano-aterials as electrochemical detectors in miniaturized system and

apillary electrophoresis was reviewed [161]. This article men-ioned three main types of NPs, which are carbon nanotubes (CNTs),Ps and nanorods, in various designs as well as the benefit of usingifferent NPs.

Compared to metal-NPs, it seems to be that CNTs have beensed in higher extension. Owing to the general superb propertiesf NPs over the bulk materials in terms of the greater surface areahat affect on the lower overpotential and higher sensitivity. Espe-ially, for CNTs detector, they provided the higher stability andesistance to passivation [161], this characteristic implies bettereproducibility because the resulting signal lacks of surface fouling.herefore, CNTs is one of the popular material used as detector inoth conventional and microfluidic miniaturized system. For exam-le, Escarpa and coworkers have reported the use of microchipapillary electrophoresis with CNTs for the determination of water-oluble vitamins [162]. It is clearly showed that the analyticalensitivity was enhanced ranging from 4 to 16 times by using theNTs modified screen-printed carbon electrode. The analysis timeas only 400 s. Using the adaptation of integration concept of cal-

bration and quantitative analysis, a protocol that combined theample analysis and the methodological calibration is presentedn Fig. 8. This is very useful for applying the present method for

uality control purpose. Moreover, this research group was alsoreated a new generation of simple microfluidic chip for analyticalomain based on a dual format of the single-channel chips withNTs detectors for fast detection of antioxidant [163]. The ultimateoal of working is similar to previous work [162] in order to obtain

ica Acta 690 (2011) 10–25 23

the ultra fast determination and improve the sensitivity in complexsamples.

Currently, on-chip immunoassays are attracting a considerablelevel of attention and have been demonstrated to be highly effi-cient tools for conducting a diverse array of immunoassays. Thecombined use of an electro-microchip, a nanogold probe, and sil-ver enhancement in an immunoassay with the readout based uponthe detection of the impedance change, has been developed [164].Here, the AuNPs were introduced into the electro-microchannel bythe specific binding between the antibodies and nanogold conju-gates and were then coupled with a silver enhancer. The “bridge”between the two electrodes of the electro-microchip was con-structed by the precipitation of silver, allowing electrons to flow.As formatted, this electro-microchip offered high detection sen-sitivities. In addition, the assays were rapid and required fewersteps than the conventional methodology. Using IgG and proteinA as substrates, detection levels of 10 �g mL−1 and 1 ng mL−1 wereobtained, respectively. Thus, the results obtained show that thisapproach is likely to have many potential uses in protein microarrayresearch and clinical diagnosis.

Even though there are only a few publications describing theuse of NPs as detectors in miniaturized devices, it is clear thattheir use typically yields improved sensitivity and a lower detectionlimit (threshold). Therefore, a variety of NP-based sensors wouldimprove a diverse array of applications. To support the capabili-ties of this important analytical technique, Escarpa’s group havereviewed the state of art of the analysis of real samples usingmicrofluidic devices [165,166]. These articles contain the criticallydemonstrated and discussed with the respect to the strengths andpuniness found in the applications of clinical, environmental andfood analysis.

4. Conclusions

During recent decades, the high potential of metal NPs in bio-analytical applications has been demonstrated and has led to thenumerous publications and advances. This review explores thepotential and advantageous features of this approach, highlight-ing the constructing of sensors and/or biosensors that exhibitan enhanced performance with respect to previous designs. Dif-ferent nanomaterials have been successfully applied to variousbio-applications. The unique properties of metal NPs include theimmobilization of biomolecules, retaining their biological activ-ity, and the formation of an efficient conducting interface withelectrocatalytic ability. In addition, they are a powerful tool tomodify the electrode materials and to construct robust and sensi-tive biosensors, which have applications in many fields of interest.Nevertheless, for miniaturized systems, there are very few applica-tions that use metal NPs in the detection schemes. This is expectedbecause of the maturity of the technology is still in its early periods.However, by taking into account the high performance in sep-aration of miniaturization and high sensitive of electrochemicaldetection, the need for coupled both two techniques is of majorimportance in view of the growing trend towards miniaturizedassays. Therefore, the fabrication of a new platform of miniatur-ized devices for bioanalysis is a great challenge that will likely beapplied in diverse fields, including clinical diagnosis, food analysis,process control and environmental monitoring, in the near future.

Acknowledgements

The authors would like to thank the Thailand Research Fund,the Department of Chemistry, Faculty of Science, ChulalongkornUniversity, the Thai Government Stimulus Package 2 (TKK2555),under the Project for Establishment of Comprehensive Center for

2 a Chim

IUlSuf

R

4 W. Siangproh et al. / Analytic

nnovative Food, Health Products and Agriculture, Chulalongkornniversity for financial support, PCU100.2010 for editing the

anguage and the Department of Chemistry, Faculty of Science,rinakharinwirot University and the Department of Chemistry, Fac-lty of Science, King Mongkut’s University of Technology Thonburior collaboration.

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