Gold and silver nanoparticles for clinical diagnostics — From genomics to proteomics

J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 2 8 1 1 – 2 8 2 3

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Review

Gold and silver nanoparticles for clinical diagnostics — Fromgenomics to proteomics☆

Miguel Larguinhoa, b, Pedro V. Baptistaa,⁎aCIGMH/DCV, Faculdade de Ciências e Tecnologia - Universidade Nova de Lisboa, Campus da Caparica, 2829-516 Caparica, PortugalbBioScope Group, Physical Chemistry Department, Faculty of Sciences of Ourense, University of Vigo, 32004, Ourense, Spain

A R T I C L E I N F O

This article is part of a Special Issue entit⁎ Corresponding author. Tel./Fax: +351 212 94

E-mail address: [email protected] (P.V. Bap

1874-3919/$ – see front matter © 2011 Elseviedoi:10.1016/j.jprot.2011.11.007

A B S T R A C T

Keywords:
Nanotechnology has prompted researchers to develop new and improved materials aimed atbiomedical applications with particular emphasis in diagnostics and therapy. Special interesthas been directed at providing enhanced biomolecular diagnostics, including SNP detectiongene expression profiles and biomarker characterisation. These strategies have focused on thedevelopment of nanoscale devices and platforms that can be used for single moleculecharacterisation of nucleic acid, DNA or RNA, and protein at an increased rate when comparedto traditional techniques. Also, several advances have been reported on DNA analysis in realtime, at both high resolution and very high throughputs, suitable for biomedical diagnostics.Here, we shall provide a review of available nanotechnology-based platforms for biomolecularrecognition, and their application tomolecular diagnostics and genome analysis, with emphasison the use of noblemetal nanoparticles for simple and specific analysis systems. Particular focuswill be put on those already being translated into clinical settings. This article is part of a SpecialIssue entitled: Proteomics: The clinical link.
© 2011 Elsevier B.V. All rights reserved.

Genome screeningSNPsProtein biomarkersTranslation into clinicsGold nanoparticlesSilver nanoparticles

Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28122. Noble metal nanoparticles for sequence analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2812
2.1. Noble metal nanoparticles for colorimetric nucleic acid characterisation . . . . . . . . . . . . . . . . . . . . 28122.2. Noble metal nanoparticles for signal enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2813

2.2.1.Fluorescence assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28132.2.2.Luminescence assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28152.2.3.Spectral labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2816

2.3. Noble metal nanoparticles in electrochemical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28162.4. Gold/silver bimetallic nanostructures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2817

3. Noble metal nanoparticles for protein bioassays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28184. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2818Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2820References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2820

☆
led: Proteomics: The clinical link.8 530.tista).
r B.V. All rights reserved.

http://dx.doi.org/10.1016/j.jprot.2011.11.007

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2812 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 2 8 1 1 – 2 8 2 3

1. Introduction

Genome sequence analysis provides valuable informationabout the existing variations at the genotype level, which ulti-mately may result in differences in the phenotype. Some ofthese variations may influence the way a given transcript isexpressed or processed and the specific characteristics of theresulting protein, such as protein/enzyme structure, andtherefore its functionality. Genome sequence analysis canthus be used for purposes of identification and characterisa-tion of organisms and individuals, or towards understandinggenome function. For example, since completion of theHuman genome sequencing efforts, enormous amounts ofdata on genetic variations (polymorphisms) have been uncov-ered [1]. There are several types of polymorphisms in thehuman genome, (e.g. insertions and/or deletions of one ormore bases, duplications) but Single Nucleotide Polymor-phisms (SNPs) are the most frequent (68%) [2,3]. Consequent-ly, unprecedented opportunities have been created to studyand to understand the consequences of genetic variations,and to integrate new genetic information into proceduresthat can be implemented in rapid, cost effective and reliablemethods to genotype, phenotype and identify gene function.The individual genetic variability as a consequence of SNPshas been associated with individual susceptibility to severalmultifactorial diseases such as cancer [4,5], diabetes [6], andalso with individual response to therapeutics [7]. Similarly,nucleotide sequence screening has paved the way for the de-velopment of robust and widespread molecular diagnosticsplatforms that assist in the detection of pathogen agents,such as bacteria, viruses, parasites [8,9]. Molecular diagnosticsrequires highly paralleled and miniaturised assays capable ofincorporating the vast information made available by the ge-nome sequencing and genome analysis projects. Also, mostdiagnostic methods focus on detection of the response mech-anisms of disease [10] (e.g. antibody produced by the organismin response to cellular transformation; protein fragment re-leased into the bloodstream as a consequence of physiologicaltransformation). The majority of current diagnostic ap-proaches rely on the identification and quantitation of diseasebiomarkers, where proteins have a decisive and prominentrole. Some of these methods are slow and inefficient as theyonly detect a disease after the disease onset. Alternatively,technologies based on nucleic acid characterisation mayallow detection of biomarkers before the onset of disease, pre-dict a specific condition and/or evaluate risk and predisposi-tion to a given abnormal phenotype.

Over the past couple of decades, noble metal nanoparticles(NPs) have been the subject of intense research for use in bio-medicine, namely for low-cost high-sensitivity approaches formolecular recognition assays. Special attention has been paidto the development of assays and biosensing platforms capableof specific identification of nucleic acid sequences that can beintegrated into genome screening strategies and identificationof sequence polymorphisms associated to relevant phenotypes,or capable of characterising specific protein profiles of disease.In particular, gold nanoparticles (AuNPs), also referred to as col-loidal gold, possess some astounding optical and physical prop-erties for enhanced capabilities in promising tools for medical

applications, such asmore sensitive, faster, and simpler assays,which are also cost-effective. Noble metal nanoparticles have,thus, been proposed as suitable and advantageous systems tosubstitute currently established technologies for genomescreenings that rely on the multiplex capability provided byfluorescence or chemiluminescence based detection [8,11].

In this review, we will focus on the use of noble metalnanoparticles, particularly gold and silver, for the designmethodologies and their application for nucleotide sequenceanalysis and protein biomarkers, and the clinical relevanceof such applications.

2. Noble metal nanoparticles for sequenceanalysis

The emergence of nanotechnology has prompted an intenseresearch effort on the nanoscale properties of several mate-rials, such as noble metal nanoparticles. These nanoparticles,usually constituted by clustered metal atoms (3 to 107 atoms)protected by a capping agent, exhibit remarkable properties,such as highly tuneable spectral behaviour and high surfaceto volume ratios. Noteworthy are the amazing optical proper-ties – intense colour and high scattering of light – due to thelocalised surface plasmon resonance (LSPR), i.e. the collectiveoscillations of free electrons at a metal-dielectric interfacewhen the frequency of incident light coincides with the fre-quency of electron oscillation. These oscillations are influ-enced by the NPs’ size, dispersion, shape and compositionand originate the intense colours and/or very intense scatter-ing of the colloidal dispersions of NPs [12,13].

Gold nanoparticles sized between 2 and 40 nm exhibit a typ-ical LSPR band centred at around 520 nm that can be easilysynthesised via reduction of a gold salt. These AuNPs can thenbe directly functionalised with thiol-modified oligonucleotides[14], resulting inwhat is known as gold-nanoprobes (Au-nanop-robes) that can be used in amultitude of detection strategies forrecognition of specific DNA/RNA sequences [15,16]. There areconsiderably fewer reports on the use of silver NPs (AgNPs)functionalised with oligonucleotides for nucleic acid sequencedetection in comparison to AuNPs. The synthesis of AgNPswith homogeneous size distribution is considerably more diffi-cult than that of AuNPs and the efficiency of thiol functionalisa-tion is significantly lower. Despite the higher scatteringproperties of AgNPs, they do not exhibit the same intense col-ours when in solution as it is observable for AuNPs, and there-fore their application in colorimetric assays has been residual.AgNPs have been explored for the development of protocols in-volving fluorescence detection - silver enhancement [17],surface-enhanced Raman scattering - SERS [18] and in combina-tion with gold to form bimetallic NPs, with distinct assemblyconcepts - core/shell and alloy [19,20].

2.1. Noble metal nanoparticles for colorimetric nucleic acidcharacterisation

As mentioned earlier, a colloidal solution of dispersed AuNPsshows an intense red colour due to the LSPR band in the visi-ble region. Changes to the medium dielectric (such as an

2813J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 2 8 1 1 – 2 8 2 3

increase in ionic strength) cause the AuNPs to aggregate, withconcomitant red-shift of the LSPR band and the solutions turnblue [15]. This phenomenon has been explored for the devel-opment of detection schemes where the presence/absence ofa given analyte (e.g. nucleic acid sequence) prevents or causesaggregation, thus yielding the result. The repulsion betweennegative charges of capping agents (e.g. citrate) at the surfaceof AuNPs prevents aggregation. Li and Rothberg, based on thedifference in electrostatic properties from single-strand DNA(ssDNA) to double-strand DNA (dsDNA) [21] proposed a colori-metric detection scheme where ssDNA easily adsorbs to theAuNPs’ surface and stabilises them against increasing ionicstrength; conversely, dsDNA does not adsorb to the surfacedue to electrostatic repulsion and aggregation of AuNPs occurs(see Fig. 1A). Detection of single-base mismatches was possibleby de-hybridisation of previously formed dsDNA structures(perfectly matched versus single-base-mismatched) and obser-vation of its influence on an AuNPs solution facing increasingionic strength. A similar approachwasused by Lee et al. for gen-otyping, where oligonucleotide probes perfectly complementa-ry to the target allele sequence were hybridised to PCRproducts, amplified from human DNA samples, and then du-plexes were denatured andmixed with AuNPs for SNP discrim-ination [22]. A similar strategy using peptide nucleic acid (PNA)probes to control AuNPs’ aggregationwas proposedbyKanjana-warut and Su whereby PNA serves both as hybridisation probeand aggregating agent of citrate-capped AuNPs and AgNPs, inthe absence of complementary DNA. Conversely, the presenceof complementary DNA induces PNA-DNA complex formationand prevents NP aggregation [23]. Recently, Liu et al. proposedan approach for discrimination of single-basemismatches loca-lised at various positions within the target nucleotide sequenceinvolving a probe-target hybridisation followed by a treatmentwith structure-selective nucleases [24]. If S1 nuclease is used,when a perfect matched target hybridises with the probe, theresulting duplex will be protected from degradation; whereas anon-complementary/mismatched target will be digested todeoxynucleotides monophosphate (dNMPs) that stabiliseAuNPs against salt induced aggregation. A main drawback ofthis system is limited sensitivity, requiring a PCR amplificationstep prior to detection.

In 1996, Mirkin and co-workers described the use of oligo-nucleotide functionalised AuNPs for specific DNA sequenceanalysis based on the colour change of Au-nanoprobes solu-tion upon hybridisation to a complementary target [14,25,26].Upon hybridisation, the target sequences and the nanoprobesform a network, the AuNPs are brought into close vicinity andaggregation is induced, resulting in a change of colour fromred to blue - see Fig. 1B. This system has also been used in amicroarray format, where a capture probe is functionalisedto the surface of the array and the second probe (functiona-lised with an AuNP) hybridises to the captured target. Follow-ing capture and hybridisation, the Au-nanoprobe is coatedwith silver via a localised reduction of silver, thus amplifyingdetection through coupled evanescent wave-induced lightscattering. This very system was used for SNP genotyping as-sociated with thrombotic disorders on unamplified humangenomic DNA [27].

Using only one Au-nanoprobe in a non-cross-linking ap-proach, Baptista and co-workers developed a sensitive

method for specific identification of nucleic acids sequences[9,15,28]. Upon hybridisation, an increase in ionic strengthwill cause extensive aggregation and concomitant change ofthe solution from red to blue; this aggregation is preventedin case the Au-nanoprobe hybridises specifically to the com-plementary target (Fig. 1C). This method is capable of detect-ing single base mismatches (mutations or single nucleotidepolymorphism, SNP) at room temperature [29], provided opti-mal conditions are met i) better discrimination is attainedwhen the mismatch is localised at the 3'end of the nanoprobesequence; ii) probe oligonucleotides on the AuNPs’ surfacestrongly influences hybridisation, thus Au-nanoprobe densityshould not be higher than 24 pmol/cm2 so as not to induce re-pulsion of the target sequence due to the phosphate backbone[30]. An overview of AuNP-based biomolecular assays for de-tection of single base mismatches is presented in Table 1.

Conversely to what has been described for AuNPs, thereare only a few reports on the use of silver nanoparticles forcolorimetric detection of nucleic acids, probably due to thelower efficiency of surface functionalisation provided by sil-ver. AgNPs have been functionalised with oligonucleotides(Ag-nanoprobes) in an attempt to increase sensitivity due tothe favourable optical properties of silver [20]. Thompson etal. proposed a cross-linking approach involving two Ag-nanoprobes, each complementary to half of the target se-quence used in a similar way to that of the cross-linkingmethod described for AuNPs [31]. An interesting approachusing naked AgNPs for detection of specific sequencesthrough non-covalent interactions between oligonucleotidesand AgNPs surface has been reported by Xu et al. [32]. In thepresence of high ionic strength, AgNP aggregation (and con-comitant colour change of solution) can be prevented by theunspecific adsorption of free oligonucleotides. This approachwas designedmainly for poly(A) sequence analysis, benefitingfrom its affinity towards the coralyne ligand that bindsstrongly to adenine-rich regions. Recent reports havehighlighted poly(A) sequences as putative new therapeutictargets for RNA-based drug design [33]. In the absence of cor-alyne, poly(A) targets adsorb non-specifically to the AgNPs’surface, stabilising them against an increase in ionic strength.When coralyne is present in solution, it binds the poly(A) tar-gets forming a duplex that prevents the oligonucleotides frominteracting and stabilising the AgNPs, and aggregation occurs.Also, a colorimetric assay for characterisation of DNA:proteininteractions based on DNA-AgNPs conjugates has been pro-posed for assessing the function of Oestrogen receptor alpha(ERα), a nuclear receptor which modulates oestrogen produc-tion through regulation of transcription. Increasing ERα con-centration helped stabilisation of Ag-nanoprobe against highionic strength [34]. In this study, Ag-nanoprobes were shownto be more sensitive than their gold counterparts.

2.2. Noble metal nanoparticles for signal enhancement

2.2.1. Fluorescence assaysApplications of AgNPs to metal-enhanced fluorescence (MEF)for DNA hybridisation assays have also been reported(Table 2), profiting from the enhancement of fluorescencewhen fluorophores are in the vicinity of noble metal nanos-tructures — see Fig. 2. AgNPs have been successfully utilised

Fig. 1 – Gold nanoparticle based colorimetric assays. (A) colorimetric assay based on naked gold nanoparticles (AuNPs) – thepresence of ssDNA stabilises AuNPs against salt induced aggregation, whereas double-strand DNA does not; and(B)cross-linking hybridisation assay — hybridisation brings both Au-nanoprobes in close vicinity leading to aggregation andconcomitant colour change; (B) non-cross-linking hybridisation assay - an increase in ionic strength causes Au-nanoprobeaggregation (blue solution), which is prevented by the presence of the complementary target.


in a MEF-based system in a microarray platform to improvesensitivity [35]. Li et al., using a flat silicon surface (p-Chips)functionalised with AgNP-films, tested the fluorescence en-hancement on four different fluorophores, demonstratingthe potential of MEF-based detection for multiplex sequenceanalysis [17]. MEF-based RNA sensing has also been described

using AgNP-films on glass surfaces and immobilisedthiolated-oligonucleotide probes [36]. First, a fluorophore-labelled oligonucleotide was hybridised to the RNA, and thisconjugate was then hybridised to the immobilised probeon the surface, thus bringing the fluorophore and theAgNPs close together. This method presented a 100-fold

Table 1 – An overview of gold nanoparticle-based bioassays for point mutation/SNP discrimination.

Detection Method Sample / Application Refs

Colorimetric NakedAuNPs

AuNP stabilisation/destabilisation upon increasing ionicstrength provided by ssDNA oligonucleotide probesfollowing hybridisation to target.

Detection of single-base mismatches –SNPs and genotyping

[21,22]

PNA probes used both as hybridisation probes and asaggregating agent for AuNPs. Probe hybridisation tocomplementary target prevents AuNP aggregation.

Single-base mismatch discrimination. [23]

S1 nuclease is used to degrade probe:mismatch targetduplexes; resulting dNMPs stabilise AuNPs against salt-induced aggregation.

Detection of point mutations in viralgenomic DNA

[24]

Au-nanoprobes

Two Au-nanoprobes, each complementary to adjacenthalves of target sequence hybridise to target, inducesnanoprobe aggregation.

Detection of single-base mismatch, in-sertion and deletion; SNP genotyping ofhuman genomic DNA

[14,25–27]

Au-nanoprobe hybridisation to complementary targetresults in duplex formation at nanoprobe surface,increasing stability against salt-induced aggregation.

Detection of single-base mutation onhuman β-globin gene; SNP discrimina-tion in the DOR gene

[9,15,28–30]


improvement on RNA quantification sensitivity, in compari-son to previously developed assays. Another approach hasbeen to use microwave-accelerated metal-enhanced fluores-cence (MAMEF) in DNA hybridisation assays, with high detec-tion limits [37]. This procedure involves the use of a MEF

Table 2 – Gold and silver nanoparticle-based signal en-hancement for fluorescence, luminescence and spectros-copy detection.

Detection Method Application /Sample

Refs

Fluorescence AgNP-based metalenhancedfluorescence (MEF)

Multiplex DNAanalysis

[17,35]

RNA quantification [36]Microwave-accelerated metal-enhanced fluores-cence (MAMEF)

Identification ofspecific nucleotidesequences

[37]

Luminescence AuNP-catalysedsilver enhancement

Specific sequenceidentification andquantification;BRCA1 mutations

[38,39]

Ag+ release uponAgNP dissolutionforchemiluminescencedetection

SNP identification [40]

Photoluminescenceof AgNPs

Detection of single-base mutation asso-ciated to sickle-cellanaemia

[41]

Spectroscopy AuNP-enhancedSurface-enhancedRaman scattering(SERS)

Detection of viralgenomic DNA

[42,43]

Identification ofsingle-basemismatches

[44]

Detection of humanBRCA1 splicevariants

[45]

AgNP-enhancedSurface-enhancedRaman scattering(SERS)

SNP in KRASoncogene

[18]

Identification ofnucleotidesequences

[46,47]

scheme in which low-power microwave heating is used inorder to promote biomolecular recognition events.

2.2.2. Luminescence assaysApproaches for AuNP-based labelling and chemiluminescence-based detection exhibiting good sensitivity, sequence specificdiscrimination and DNA target quantification have also beenreported (see Table 2), in particular microarray platformscoupled to AuNP-catalysed silver enhancement [38,39]. A suc-cessful genotyping approach targeting the BRCA1 gene wasreported by Girigoswami and co-workers, which includes twoprimers (primer1 and primer2), each complementary to half ofthe target sequence [39]. Primer1 bears a 5’ zip complementar-ity, necessary for analysis to be carried on a zip-code microar-ray platform. Primer2 is 3’ thiol-modified, required to bind anAuNP for silver enhancement-based detection. Upon successfulhybridisation, a ligase reaction is carried out, to seal the nickbetween the two primers. When the allele-specific probe is100% complementary to the target, ligation occurs and, uponassembly on the microarray platform, the sequence binds anAuNP through 3’-thiol modification. Afterwards, the AuNP issilver-coated, for enhanced detection.

A chemiluminescent approach for DNA detection was de-scribed, whereby the Ag+ ions arising from AgNPs dissolutionact as indicators for detection [40]. In this system, two differ-ent probes are used, complementary to different halves ofthe target, similar to the cross-linking method previously de-scribed (see above). Following successful probe-target hybridi-sation, the AgNPs are dissolved using HNO3 and Ag+ ionsdetermined via a coupling chemiluminescence reaction.SNPs were analysed by comparison of thermal dissociationcurves for perfect complementary targets and mismatchedtargets, hybridised with the referred probes with very lowlimits-of-detection. Guo et al. proposed another approach fordiscrimination of single base alterations based on oligonucle-otide probes with a cytosine loop (C6) and AgNPs’ photolumi-nescence detection, and used this method to discriminate asingle-base mutation associated to sickle-cell anaemia [41].Upon probe hybridisation to a perfect complementary target,an efficient scaffold of Ag+ ions originates a photoluminescentAgNP, whose emissive properties potentiate the detection.Mismatched targets yielded residual luminescence emission.

Fig. 2 – Schematic configuration for a metal-enhanced fluorescence (MEF) system. Fluorescence signal enhancement may beachieved by deposition of AgNPs onto a surface, before probe immobilisation. Upon hybridisation with fluorophore labelledprobe, the intensity of fluorescence emission is potentiated by the AgNPs (MEF).


2.2.3. Spectral labelsAu- and AgNPs have also been used as signal enhancers forRaman scattering-based detection of biomolecules [18].AuNPs for Raman spectroscopy-based detection have beenused in combination with i) methylene blue for target label-ling [42], ii) magnetic nanoparticles for effective target capture[43], and iii) assembled on a multi-metal nanojunction struc-ture for identification of single-base mismatches in an HIV-1-derived DNA sequence [44]. SERS detection potentiated byAuNPs was further used on cancer gene expression studiesaiming at detection of multiple splice variants of humanBRCA1 gene analysis [45]. Fig. 3 illustrates a possible approachfor SERS signal enhancement, using AuNPs as spectral labels.

AgNPs have also been used as Raman scattering enhancersfor nucleotide sequence analysis (see Table 2). Zhang et al. pre-sented an approach, using a combination of mixed Ag-nanoprobes and Raman reporter molecules, for multiplex de-tection of specific nucleotide sequences, through a sandwichhybridisation assay for recognition and signal amplification[46]. Recently, Lo and co-workers used AgNPs for enhancementof the Raman scattering signal, using an Au-nanotip coveredwith oligonucleotide molecules and the subsequent depositionof sub ~10 nm AgNPs to coat the Au-nanotip [47]. A distinctmethod for SNP genotyping based on a ligation reactioncoupled to Raman scattering detection and including twoprobes (each complementary to different halves of a target se-quence) was presented by Lowe et al. [18]. A first oligonucleo-tide probe hybridises with the target strand downstream of aSNP and is later conjugated to an AgNP for signal amplification.The second probe, containing a Raman active fluorophore and

a discriminating base at the 3′ end, binds upstream of theSNP and adjacent to the first probe allowing maximum speci-ficity. In presence of a perfect match, ligation between thetwo oligonucleotides is attained, bringing the fluorophore andthe AgNP closer. This enhances the signal, and particularRaman signatures may be observed as different fluorophoresare placed on the SNP allelic specific probes. This approach al-lows for flawless discrimination of SNPs occurring in the KRASoncogene at picomolar detection levels.

2.3. Noble metal nanoparticles in electrochemical methods

Electrochemical genosensors usually rely on modification ofan electrode surface with ssDNA oligonucleotide strands, as-sembled into a monolayer, and characterisation of the samesurface using electrochemical techniques [48]. Differenttypes of electrodes (e.g. gold, glassy carbon, etc.) have beencombined with AuNP-based protocols with or without silverenhancement or electrode surface modification – see examplein schematic in Fig. 4. Pan et al. used AuNPs@biotin-streptavi-din conjugates for characterisation of specific sequencesrecognised by DNA-binding transcription factors [49]. Au-Fe3O4 magnetic composites with silver enhancement havebeen used (as an alternative to AuNPs as labels) to detect a27-mer DNA target using a glassy carbon electrode as surface[50]. Noor et al. proposed a sensor consisting of an immobi-lised probe arranged in a hairpin conformation that is dis-rupted upon hybridisation with a perfect complementarytarget, which is detected by an Au-nanoprobe and used for de-tection of single base mutations in the KRAS oncogene [51].

image of Fig.�2

Fig. 3 – Signal enhancement on a surface-enhanced Raman scattering (SERS) spectrum using noblemetal nanoparticles. AuNPsused as spectral labels for hybridisation assay on a gold surface create a Raman environment around the duplexes, whichleads to increased intensity in the Raman spectrum.


Kerman et al. were able to discriminate SNPs and identify theintervening DNA bases, by combining AuNPs andapolymerasebased strategy [52], andwave-voltammetry was used tomonitorthe electrochemical signals and to pinpoint which nucleobaseswere involved in a particular SNP, namely from the tumour ne-crosis factor gene (TNF-α). Recently, differential pulse voltam-metry was used in combination with a nanoporous surface toidentify a ssDNA target, whereby nanopore blockage is mea-sured with and without successful probe-target hybridisationusing the redox indicator [Fe(CN)6]4-/3- [53]. AuNPs are employedas molecular tags, to enhance nanopore blockage, attaininglower limits-of-detection. Another type of surface nanostructur-ing was described, using a glassy carbon electrode and subse-quent depositions of AuNPs, followed by immobilisation ofoligonucleotide probes on the electrode surface. This methodwas used for identification of a specific sequence within a PCR-product amplified from the LTA-α gene [54].

AgNPsmay also be utilised for enhancement of the electro-chemical signal or as an indicator of successful hybridisation

Fig. 4 – AuNPs for surface modification of electrodes. Electrode suelectrochemical signal.

events. Niu et al. studied the interactions between luteolincopper(II) and dsDNA using cyclic voltammetry [55], and acombination of polysaccharide molecules with AgNPs as la-bels was shown by Kong et al. [56]. A different system wasdemonstrated by Rijiravanich et al. [57] where pH-controllable polyelectrolyte shells are used as encapsulatingagents for AgNPs in conjunction with the biotin-avidin com-plex for DNA labelling and detection of successful probe-target hybridisation. Afterwards, acid dissolution producessilver ions from the AgNPs, and analysis is carried by anodicstripping voltammetry and linear sweep voltammetry. Agreat advantage of this system is its application to screenprinted electrodes (SPEs), instead of conventional ones, withgood sensitivity (detection at the femtomolar level).

2.4. Gold/silver bimetallic nanostructures

Much work has been carried out on bimetallic gold/silver NPs,mainly on synthesis and characterisation [19,20,58]. Gold-

rface modified with gold nanoparticles enhancing the

image of Fig.�3

image of Fig.�4


silver NPs were firstly synthesised in an effort to combine theoptical properties of silver, namely a high extinction coeffi-cient, with the affinity of gold to bind to sulphur atoms. Forbiosensing purposes, this would result in improved sensitivitywithout risking the efficiency of functionalisation. Gold/silverbimetallic NPs may be assembled into different organisationstructures e.g. core/shell [58] and alloy [20], depending on thesynthesis procedure. Cao et al. firstly described the applica-tion of bimetallic NPs for identification of nucleotide se-quences [59]. The authors synthesised core/shell NPscomposed by a silver core and a gold shell (Ag/Au) and func-tionalised it using thiol-modified oligonucleotides, whichwere applied in a cross-linking hybridisation approach to suc-cessfully identify a fully complementary DNA target. Thesame group later reported a dual-colour system includingAu-nanoprobes and Ag/Au core/shell-nanoprobes for the de-tection of a 30-mer specific nucleotide sequence correspond-ing to a portion of the human beta-globin gene anddiscrimination of the SNP responsible for sickle-cell anaemia[60] — see Fig. 5A.

Contrary to what happens in the laborious methods forsynthesis of core/shell NPs, gold/silver alloy NPs (AuAg-alloy-NPs) are formed by co-reduction of both gold and silverions, by the same reducing agent, in a much simpler andrapid process. Another advantage is the possibility of SPRband modulation, according to the gold:silver ratio [20]. Re-cently Doria et al. reported a proof-of-concept consisting onAuAg-alloy NPs functionalised with oligonucleotide probes(AuAg-alloy nanoprobes) for identification of nucleotide se-quences, using a non-cross-linking hybridisation assay [61].The authors verified that AuAg-alloy nanoprobes were com-parable to Au-nanoprobes for nucleic acid sequence analysisand further proposed an approach where AuAg-alloy nanop-robes and Au-nanoprobes are used for simultaneous detec-tion of multiple target sequences, in a one-pot dual coloursystem (Fig. 5B) for identification of gene sequences derivedfrom tumour suppressor gene TP53 and proto-oncogene c-MYC. Besides this work, a recent report describes annealingof Au/Ag-alloy nanostructures on a glass surface, coatedwith a silicon:carbon nanometric film and modified with anoligonucleotide probe, where DNA probe:target hybridisationwas monitored by MEF [62].

3. Noble metal nanoparticles forprotein bioassays

Most clinical diagnostics applications strongly rely on the identi-fication of biomarker proteins for disease characterisation, andan enormous effort has been put into developingnanotechnology-based approaches [63,64]. Several platformsspecifically designed for protein biosensing often include stan-dard assembly concepts using antigens and antibodies for mo-lecular recognition (e.g. sandwich immunoassay), coupled todistinct detection strategies, such as spectroscopy [65–68], elec-trochemistry [69–71] or imaging [72–74]. Noble metal nanoparti-cles have brought a new dimension to such bioassays byincreasing the sensitivity at lower costs [64,70]. Because mostof these assays rely on previously studied and widely used

biomarkers, integration into nanoparticle-based sensing plat-forms has been rather straightforward – Table 3.

Most gold nanoparticles applications have been directedtowards signal enhancement of standard protein detectionassays, ranging from enzyme-linked immunosorbent assays(ELISAs) to immunoassay platforms coupled to electrochemi-cal or SERS-based detection. Examples include increase inRayleigh scattering intensity for Alzheimer disease diagnos-tics [65], nanochannel-based filtering and sensing platformfor cancer biomarkers [70] and enhancement for immuno-chromatographic test strips [84]. Resonance Rayleigh scatter-ing was used in conjunction with antibody-coated AuNPs fortransferrin biosensing [66] for transferrin quantification inhuman serum samples with a detection limit two orders ofmagnitude lower than ELISA. A similar concept was shownfor two-photon Rayleigh spectroscopy detection of tau proteinin cerebrospinal fluid. By using monoclonal anti-tau anti-bodies functionalised to AuNPs’ surface, it was possible to im-prove scattering intensity by 16-fold, which reflects asignificant increase in sensitivity (by two orders of magnitude)[65]. Femtomolar levels of prostate-specific antigen (PSA) inserum were detected using an immunoassay coupled toSERS-based detection. Monoclonal antibodies were usedin this assay, with the secondary antibody being directlyattached to a multifunctionalised AuNP [67]. A differentplatform using a naked-eye detection method and consist-ing of a AuNP-based enhancement for immunochromato-graphic test strips showed promising results for clinicaldiagnostics purposes [84]. In this sandwich-like assay,both the primary and the secondary antibodies are conju-gated with AuNPs, increasing the limit of detection of thechorionic gonadotropin hormone (HCG) in human serumby 1 order of magnitude to reach 10 pg/mL and of thetotal PSA to reach 200 pg/mL. Besides the advantage in sen-sitivity, this is a simple and fast method which providesresults in less than 20 minutes. Self-assembled gold col-loids functionalised with extractable nuclear antigens(ENAs) have also been used in combination with an opticalfibre evanescent-wave sensor, for anti-nuclear antibody(ANA) detection in human serum [85]. This system doesnot require a secondary antibody and presents increasedsensitivity by an order of magnitude when compared to tra-ditional ELISA.

AuNPs have also been utilised as optical signal enhancerswhen used in conjunction with the traditional ELISA test [83].The signalling antibody (secondary antibody conjugated to a re-porter molecule) was attached to the surface of AuNPs and theoptical signal resulting from molecular recognition was thencompared in the presence and absence of the AuNP-antibodyconjugates. Besides the verified 2-fold increase in sensitivity,the use of AuNPs resulted in shorter incubation times in orderto obtain a colorimetric result for detection of the breast tumourbiomarker CA15-3 at clinically relevant levels.

4. Conclusions and future perspectives

Several systems based on noble metal NPs, namely gold and sil-ver, have been proposed that are capable of nucleic acid se-quence (e.g. SNPs discrimination) and protein characterisation,

Fig. 5 – Gold:silver nanoprobes for colorimetric assays. (A) two-colour detection scheme via a cross-linking hybridisation assayusing AgAu-core/shell-nanoprobes – upon hybridisation, the nanoprobes and are brought in close vicinity, leading toaggregation and concomitant colour change; and (B) dual-colour system using gold-nanoprobes and gold-silver-alloynanoprobes in a non-cross-linking format.


and suitable for clinical application. The signal enhancementthat noble metal nanoparticles bring into protein detectionschemes has enabled for the development of extremely sensi-tive diagnostic platforms for early detection of disease

biomarkers of clinical relevance. These nanoparticles havefound application in diverse strategies, such as electrochemis-try, luminescence, target labelling, SPR-based biosensors, andmay further be combined in different assembly structures, to

image of Fig.�5

Table 3 – Protein bioassays based on noble metal nanoparticles — a clinical perspective.

Category Detection technique Target / Clinical Application Ref.

Scanometric Light scattering Human chorionic gonadotropin (HGC), Prostate-specific antigen (PSA) andα-fetoprotein (AFP) as cancer biomarkers

[75,76]

Spectroscopy Rayleigh scattering Tau protein and human holotransferrin; PSA as cancer biomarker [65,66,77]SERS Thrombin, PSA and carcinoembryonic antigen (CEA) as cancer biomarkers;

prion protein PrPc[67,68,78,79]

UV-Vis spectroscopy (naked-eye detection)

Cyclic A2 as a cancer biomarker; Glycated haemoglobin (HbA1c) as diabetesmarker

[80,81]

Immunoassay Dynamic light scattering CA152, CEA, CA19-9 and PAP (prostatic acid phosphatase) as cancerbiomarkers

[82]

ELISA CA15-3 cancer biomarker [83]Immunocromatography(naked-eye detection)

HCG and PSA as cancer biomarkers [84]

Gold-modified optic fiber Anti-nuclear antibodies (ANA) [85]UV-Vis spectroscopy PSA and CEA as cancer biomarkers [86,87]

Electrochemicalimmunoassay

Amperometry Interleukin-6 (IL-6) and PSA as cancer biomarkers [69,88]Differential pulsevoltammetry

CA15-3, tumour necrosis factor alpha (TNF-α) and Hepatitis B surfaceantigen

[70,89,90]

Square wave voltammetry Human serum albumin and cardiac myoglobin as biomarkers; HIV-1 reversetranscriptase level in human serum

[91–93]

Cyclic voltammetry CEA and human cardiac troponin I as cancer biomarkers [71,94]Imaging Surface plasmon resonance

imagingEpidermal growth factor receptor (EGFR) as cancer biomarker [72]

SERS imaging Human epidermal growth factor 2 (HER2) as cancer biomarker [73]Reflectance imaging Prostate-specific membrane antigen (PMSA) as a cancer biomarker [74]


promote synergistic conditions with concomitant increase insensitivity and versatility. Most of the current platforms are fo-cused on increasing sensitivity and/or throughput ratio but stillrely on highly intensive, specialised and laborious technicalinput for sample treatment. Thus far, most of the reported con-cepts still need validation in real human samples and/or clinicalsettings. Most applications of nanoparticle based approacheshave been demonstrated in simpler conditions using syntheticoligonucleotides as targets, microorganisms and/or viruses,and well characterised protein biomarkers, which, though rele-vant for conceptualisation, are still far away from the severestrains of clinical screening strategies. Once current applicationsare refined, the developed platforms may easily be translatedfrom proof-of-concept to clinical setting, paving the way fromlow throughput genotyping approaches to more robust plat-forms capable of multiplexing and medium to high throughputscreening for wide genome and proteome characterisation.

Acknowledgements

The authors acknowledge FCT/MCTES (Portugal) for financialsupport for CIGMH and SFRH/BD/64026/2009 grants for M.Larguinho.

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