Analytical Biochemistry 417 (2011) 116

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Review

Gold nanoparticle-based signal amplication for biosensing

Xiaodong Cao a, Yongkang Ye b, Songqin Liu a,
a
State Key Laboratory of Bioelectronics, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China
b
School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, China

a r t i c l e i n f o a b s t r a c t

Article history: Colloidal gold nanoparticles (AuNPs), with unique properties such as highly resonant particle plasmons,
Received 4 March 2011 direct visualization of single nanoclusters by scattering of light, catalytic size enhancement by silver
Received in revised form 9 May 2011 deposition, conductivity, and electrochemical properties, are very attractive materials for several applica-
Accepted 17 May 2011
tions in biotechnology. Furthermore, as excellent biological tags, AuNPs can be easily conjugated with
Available online 26 May 2011
biomolecules and retain the biochemical activity of the tagged biomolecules, making AuNPs ideal trans-
ducers for several biorecognition applications. The goal of this article is to review recent advances of
Keywords:
using AuNPs as labels for signal amplication in biosensing applications. We focus on the signal ampli-
Gold nanoparticle
Amplication
cation strategies of AuNPs in biosensing/biorecognition, more specically, on the main optical and elec-
Recognition trochemical detection methods that involve AuNP-based biosensing. Particular attention is given to
Biosensing recent advances and trends in sensing applications.
2011 Elsevier Inc. All rights reserved.

Achieving high sensitivity is one of the major goals in biological
assays such as the monitoring of protein (enzymes, antigens, and
antibodies) interactions and nucleic acidnucleic acid/protein
hybridization events. Many efforts have been made toward the
exploration of novel means to enhance detection sensitivity. Suc-
cessful strategies include the employment of new labels such as
electroactive molecules, redox complexes, and metal ions; the inte-
gration of enzyme-assisted signal amplication processes; and the
incorporation of nanomaterials to increase the upload of electro-
chemical tags and so on [16]. Among these approaches, the nano-
material-based one has been widely used during recent years as
versatile and sensitive tracers for the electronic, optical, and micro-
gravimetric transduction of different biomolecular recognition
events [712]. The remarkable signal enhancement associated
with the use of nanoparticle amplication labels and the formation
of nanoparticlebiomolecule assemblies provides the basis for
Corresponding author. Fax: +86 25 52090618.
E-mail address: liusq@seu.edu.cn (S. Liu).
ultrasensitive optical and electrical detections with polymerase
chain reaction (PCR)1-like sensitivity.
Colloidal gold nanoparticles (AuNPs) have been widely used as
labels for analytical signal amplication and biomedical purposes
for many years [11,13,14]. AuNPs can be produced in an aqueous
solution with rapid stirring by reducing chloroauric acid (HAuCl4)
with reducing agent. The Au3+ ions are reduced to neutral gold
atoms. The solution becomes supersaturated as a result of more
gold atoms forming, and gold gradually starts to precipitate in
the form of sub-nanometer particles. The rest of the gold atoms
that formed during reduction are absorbed on the existing parti-
cles. In addition, if the solution is stirred vigorously enough, the
1
Abbreviations used: PCR, polymerase chain reaction; AuNP, gold nanoparticle;
HAuCl4, chloroauric acid; ECL, electrochemiluminescence; BSA, bovine serum albu-
min; IgG, immunoglobulin G; PPD, poly(o-phenylenediamine); Fc, ferrocene; QD,
quantum dot; HRP, horseradish peroxidase; TMB, 3,30 ,5,50 tetramethylbenzidine; P-
gp, P-glycoprotein; ConA, concanavalin A; ssDNA, single-stranded DNA; ELISA,
enzyme-linked immunosorbent assay; CL, chemiluminescent; AFP, a-fetoprotein;
ASV, anodic stripping voltammetry; HQ, hydroquinone; SERS, surface-enhanced
Raman scattering; scFv, single-chain variable fragment; HNP, Hantaan virus nucle-
ocapsid protein; FRET, Frster resonance energy transfer; Lec-QD, lectin-conjugated
QD; PL, photoluminescence; Carbo-AuNP, carbohydrate-conjugated AuNP; PRRSV,
porcine reproductive and respiratory syndrome virus; TR-FRET, time-resolved
uorescence resonance energy transfer; LSPR, localized surface plasmon resonance;
rRNA, ribosomal RNA; SPR, surface plasmon resonance; RSD, relative standard
deviation; RCA, rolling circle amplication; LSPCF, localized surface plasmon coupled
uorescence; NIR, near infrared; HSA, human serum albumin; DLS, dynamic light
scattering; PSA, prostate-specic antigen; f-PSA, free PSA; RLS, resonance light
scattering; ApoAI, apolipoprotein AI; DPV, differential pulse voltammetry; SWV,
square wave voltammetry; EIS, electrochemical impedance spectroscopy; MEA,
microelectrode array; ICP-MS, inductively coupled plasma mass spectrometry.

0003-2697/$ - see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2011.05.027
2 Gold nanoparticle-based signal amplication / X. Cao et al. / Anal. Biochem. 417 (2011) 116
particles will be fairly uniform in size. AuNPs have advantages such
as rapid and easy synthesis, narrow size distribution, efcient sur-
face modication by thiols or other bioligands, and desirable bio-
compatibility. Properties such as electron dense core, highly
resonant particle plasmons, direct visualization of single nanoclus-
ters by scattered light, catalytic size enhancement by silver depo-
sition, conductivity, and electrochemical properties have made
AuNPs to be attractive materials for several applications in bio-
technology. Furthermore, AuNPs can be easily conjugated with bio-
molecules and retain the biochemical activity of the tagged
biomolecules, leading AuNPs to be excellent transducers for sev-
eral biorecognition applications [1518]. Here we provide a com-
prehensive overview of recent advances in biosensing by using
AuNPs as labels for signal amplication. Various signal amplica-
tion routes by AuNPs in biosensing/biorecognition, specically
where AuNPs serve as carriers for capture probe, catalyst, and plat-
form for the deposition of silver to provide further signal enhance-
ment, are summarized. In addition, the main optical and
electrochemical detection techniques and methods involved in
the use of AuNPs are discussed.
Sensing/signal amplication strategies
To achieve high sensitivity for the detection of biotargets, a num-
ber of sensing/signal amplication strategies have been developed
through the use of various size/shape gold nanostructures (mostly
AuNPs) coupled with biorecognition steps such as nucleic acid
hybridization and the antibody/antigen sandwich-type procedure.
These strategies include the use of AuNPs as carriers for both probe
and signal molecule loading, as indicators to induce the deposition
of silver on AuNPs for both optical and conductivity detection, as la-
bels in coupling with bio-barcode techniques, and as receptors
when employing Frster resonance energy transfer methods.
AuNPs as carriers
Because of the narrow size distribution, good biocompatibility,
and ease of modication with thiol groups, AuNPs could serve as
carriers for the immobilization of captured probes. These applica-
tions include immobilization of more biorecognition elements or
a large number of optical or electrochemical tags on AuNPs, which
provide more binding sites or amplication of the analytical signal
in a single recognition reaction (Fig. 1) [1940], and target-induced
chemical deposition of metals [4173], especially silver [4167].
Immobilization of more active molecules on AuNPs
Wang and coworkers fabricated an AuNP layer on the surface of
gold electrode for immobilization of additional primary antibodies.
4-(Dimethylamino)butyric acid was tagged on the (anti)-analytes,
Fig.1. Signal amplication strategies based on AuNPs serving as carriers. (A) AuNPs were immobilized directly onto a solid substrate to capture more capture probes.
Sandwich-type complexes could be formed after two steps: specic recognition between capture probes and targets and targets and detection probe modied with signal
inducers. The signal amplication could be achieved, although more than one specic recognition occurred on a single AuNP. (Reformed from Ref. [20].) (B) Capture probes
were rst immobilized on a solid substrate. After the rst step, specic recognition between capture probes and targets, AuNPs carrying lots of detecting probes modied with
signal inducers were introduced to the substrate through the second step specic recognition, thereby resulting in the signal amplication. (Reformed from Ref. [30].)
which generated electrochemiluminescence (ECL) signal on con-
tact with tris(2,20 -bipyridine)ruthenium solution. Because of the
high adsorption capacity of AuNPs, 10- and 6-fold sensitivity
enhancement was obtained for bovine serum albumin (BSA) and
immunoglobulin G (IgG), respectively, over their direct immobili-
zations on the electrode [20]. This signal amplication was also
demonstrated with N-(aminobutyl)-N-ethylisoluminol as labels
[19]. Owing to more labeled protein being accumulated on AuNPs,
the sensitivity of the resulting immunosensor had 3 orders of mag-
nitude improvement over those without AuNPs as the immobiliza-
tion support. Similar signal amplication was reported by Shen and
coworkers, where a sensitive electrochemical immunoassay, based
on poly(o-phenylenediamine) (PPD) lm/AuNP amplication and
ferrocene (Fc) labels, was developed for the detection of immuno-
species [24]. Zhu and coworkers presented a nonlabeled ECL
immunosensor, based on CdSe quantum dots (QDs), for the detec-
tion of human prealbumin [22]. Two layers of AuNPs were con-
structed on the gold electrode surface for the linkage of
prealbumin antibodies. A detection limit of 1.0  1011 g ml1 for
prealbumin was obtained based on the inhibition of the ECL reac-
tion between CdSe QDs and K2S2O8 through the formation of
immunocomplexes. More recently, Lin and coworkers fabricated
a highly specic and sensitive DNA detection platform. It was
accomplished by means of more dually labeled stemloop DNA
probes, immobilized on the AuNPs to a 96-well microplate, an-
chored by a protein [21]. The hybridizations between the hairpin
DNA probes and the target DNA caused signicant conformational
changes of the probes, which forced biotin away from the surface
of AuNPs. As a result, the biotin became accessible to the strepta-
vidinhorseradish peroxidase (HRP) and the target hybridization
events could be sensitively detected via the HRP-catalyzed sub-
strate 3,30 ,5,50 -tetramethylbenzidine (TMB) with the use of a spec-
trophotometric method. Based on this signal amplication
strategy, they could detect DNA at the femtomolar level.
Zhu and coworkers designed a new strategy for the assessment of
cell surface carbohydrates and P-glycoprotein (P-gp) expression sta-
tus by quantication of the cell numbers with an electrochemical
immunoassay [26]. Due to the good biocompatibility of AuNPs and
excellent conductivity of nitrogen-doped carbon nanotubes, a novel
three-dimensional architecture was initially fabricated by a combi-
nation of nitrogen-doped carbon nanotubes, thionine, and AuNPs
via a simple layer-by-layer method (Fig. 2). This architecture pro-
vided an effective matrix for concanavalin A (ConA) binding and a
predominant capability for cell capture. Accompanied by the enzy-
matic reaction of HRP toward the oxidation of thionine by H2O2, the
proposed cytosensor showed excellent analytical performance for
the detection of HeLa cells and cell surface receptors P-gp.
Contrary to the direct immobilization of AuNPs on solid support
as an efcient matrix to increase the loading of primary analytes,
the development of novel colloidal gold labels has received
 Gold nanoparticle-based signal amplication / X. Cao et al. / Anal. Biochem. 417 (2011) 116
Fig.2. Schematic representation of the fabrication of the cell-based electrochemical enzyme-linked immunoassay. ConA, concanavalin A; Ab, antibody. (From Ref. [26].)
Fig.3. Schematic illustration of proposed DNA detection strategy. (From Ref. [28].)
increasing attention [2740]. Cui and coworkers reported an ECL
detection method for specic DNA sequences by the integration
of luminol-functionalized AuNP labels, amplication of AuNPs,
and the biotinstreptavidin reaction system [28]. The luminol-
functionalized AuNP labels were synthesized via the direct reduc-
tion of HAuCl4 by luminol [74]. Thousands of luminol molecules
were coated on the AuNPs as stabilizers [75]. This kind of lumi-
nolAuNPs could be coupled with single-stranded DNA (ssDNA)
without the loss of ECL activity and change in specicity of the
hybridization reaction. Thus, the luminol-labeled DNA signal
probes, which were associated with the residual base sequence
of the targets, were assembled on the electrode through specic
base pairing to form sandwich DNA conjugates on the modied
electrode (Fig. 3). As a result, an ultrasensitive DNA assay for detec-
tion of sequence-specic DNA was achieved with a detection limit
of 0.19 fM. Such a strategy has already been applied to an enzyme-
linked immunosorbent assay (ELISA) immunoassay. This was the
analysis of CA15-3 antigen with AuNPs as carriers for the immobi-
lization of more HRP-labeled anti-CA15-3 to achieve an amplica-
tion of the optical signal [30]. Zhang and coworkers represented an
enhanced chemiluminescent (CL) immunoassay for sensitive
determination of a-fetoprotein (AFP) [31,37]. The method made
full use of magnetic beads as bimolecular immobilization carriers
to separate target molecules as well as double-codied AuNP labels
modied with HRP-conjugated anti-AFP for signal amplication.
The formation of sandwich immunocomplexes between the mag-
netic beads and AuNP labels allowed the monitoring of the concen-
tration of AFP with high sensitivity through the HRP labels activity
toward the oxidation of luminol to produce light emittance. To fur-
ther increase the loading of signal molecules, nanogold-enwrapped
graphene nanocomposites were prepared as trace labels for clinical
immunoassays [34]. Signal amplication was achieved with enor-
mous loading of HRP-conjugated antibodies on the surface of nano-
gold-enwrapped graphene nanocomposites.
3
Ju and coworkers designed a novel dual-functionalized nanop-
robe for highly sensitive and selective in situ evaluation of carbohy-
drates on living cells by the integration of specic carbohydrate
recognition and enzymatic signal amplication of proteins on AuN-
Ps [35]. In their work, the nanoprobes were prepared by mixing HRP
and ConA directly with AuNPs. A nanoscaffold of nanohorns func-
tionalized with arginineglycineaspartic acidserine tetrapeptide
was prepared on an electrode surface for cell capture and enhance-
ment of the electrical connectivity. The electrochemical signal was
produced from the reduction of enzymatic reaction product on the
modied electrode. The enhanced sensitivity could be attributed to
the combination of dual signal amplication based on enzymatic
catalysis, high surface-to-volume ratio of AuNPs for protein loading,
and high molar ratio of enzyme to lectin on AuNPs. With this ap-
proach, they could electrochemically detect the model cells with
a detection limit down to 15 cells and monitor dynamic variation
of carbohydrate expression on living cells in response to drugs.
AuNP-induced chemical deposition of silver for signal amplication
The immunogold labeling technique was commonly used be-
cause of the high electron density of AuNPs, which exhibited as a
dark brown spot under an electron microscope and resulted in a
distinct image for labeling. In addition, this image response could
be further enhanced through immunogold silver staining, a tech-
nique by which silver particles were precipitated on the surface
of the AuNPs [4167]. Three strategies have been reported for
amplication of the detection signals of gold tracers (Fig. 4).
According to the rst strategy, after the conjugates of oligonucleo-
tide/anti-protein nanoparticle and the electrode-bound targets
formed, silver was deposited on the AuNPs to obtain an enhanced
electrochemical signal (Fig. 4A) [4249].
Inspired by the use of AuNP labeling and subsequent silver
enhancement, Wang et al. proposed a strategy for the detection
of DNA hybridization by measurement of the deposited silver with
4 Gold nanoparticle-based signal amplication / X. Cao et al. / Anal. Biochem. 417 (2011) 116

Fig.4. Detection strategies based on silver enhancement on AuNPs. (A) Voltammetric stripping assay. The hybridization event occurred between DNA strand immobilized on
the solid substrate and gold-tagged DNA. The AuNPs were rst covered with silver by a deposition treatment and then detected by stripping techniques via silver-enhanced
signal. (Adapted from Ref. [18].) (B) Conductivity assay. Probe DNA immobilized in a small gap between two electrodes was hybridized with target DNA and then with gold-
modied DNA probes. Gold was accumulated in the gap. Silver enhancement was performed in the presence of hydroquinone (HQ). The silver precipitated onto the AuNPs
improved the sensitivity of the assay by lowering the resistance across the electrode gap. (C) Scanometric assay. A surface-bound capture oligonucleotide bound one-half of
the target of interest, and an oligonucleotide-functionalized AuNP probe bound the other half. Catalytic reduction of silver onto the capture/target/probe sandwich resulted in
a signal that could be recorded with a microplate reader. (Reformed from Ref. [52].)

an extremely sensitive technique of electrochemical stripping me-
tal analysis [42]. Dramatic signal amplication was achieved by
efcient magnetic removal of noncomplementary DNA. The strat-
egy could also be employed for the detection of proteins [4449].
An electrochemical immunosensor, based on the precipitation of
silver on colloidal gold labels, has been developed. Silver metal
on dissolution in an acidic solution was indirectly determined by
anodic stripping voltammetry (ASV) at a glassy carbon electrode.
The analytical procedure consisted of the immunoreaction of the
antigen (analyte) with the primary antibody adsorbed on the walls
of a polystyrene microwell. After that, a secondary colloidal gold-
labeled antibody was bound. Then silver enhancement, acid disso-
lution, and stripping detection of the silver occurred at a glassy car-
bon electrode [44]. A high sensitivity with a detection limit of
6  1012 M human IgG was achieved. This level of detection could
be attributed to the sensitive electrochemical determination of sil-
ver ions and to the catalytic precipitation of a large number of sil-
ver ions on the colloidal gold-labeled antibody. Recently, a
sensitive magnetoimmunosensing method for antigen detection,
based on the catalytic effect of AuNPs on the electroreduction of
silver ions, was reported [47]. The authors employed micropara-
magnetic beads as primary antibody immobilization platforms
and AuNPs modied with secondary antibodies as highly sensitive
electrocatalytical labels to achieve high sensitivity. A built-in mag-
net carbon electrode allowed the local collection/immobilization of
the microparamagnetic beads with the immunological sandwich
and AuNP catalysts to attach onto the electrode surface. The selec-
tive electrocatalytic reduction of silver ions onto the surface of
AuNPs led to improved sensitivity, which resulted in a lowered
protein detection limit to 23 fg ml1. As an alternative detection
method, the silver precipitation on colloidal gold tags could be
monitored by a CL method using the Ag+K2S2O8Mn2+H3PO4
luminol system (Fig. 5) [49]. This approach could be applied to
the determination of human IgG in human serum samples with
high sensitivity and a detection limit of 3  1014 M.
The second strategy was based on the specic biological interac-
tions that occurred on a supporting surface with varied resistance or
conductance of the substrate. This method could be termed as con-
ductometric assay. Mirkin and coworkers exploited the silver deposi-
tion technique to construct a sensor based on conductivity
measurements according to the second strategy (Fig. 4B) [14]. In
their approach, the oligonucleotide capture strands were immobi-
lized in the gap (20 lm) between two electrodes. A three-compo-
nent sandwich approach was performed by hybridized target DNA
to capture AuNP-tagged reporter probes between the electrode
gaps. In the absence of target DNA, there was no current ow across
the electrode gap. However, in the presence of target DNA, the asso-
ciated nanoparticle probes and catalytically deposited silver current
could ow between the electrodes, which resulted in a sharp drop in
the resistance of the circuit. Using this method, DNA could be de-
tected with a detection limit of 500 fM. A similar approach was
developed for immunoassay by using an electro-microchip to detect
the immunoreaction signal [50]. The AuNPs were introduced into
the electro-microchip by a typical sandwiched procedure. The silver
precipitation constructed a bridge between two electrodes of the
electro-microchip, which allowed electrons to pass and led the var-
iation of resistance. The identical strategy could also be applied in
protein immunoassay [50] and bacterial biosensors [51] with an
electro-microchip transducer. The detection limits for protein A
and Acinetobacter baumannii genomic DNA sample were 1 and
0.825 ng ml1 (1.2 fM), respectively.
The third strategy (Fig. 4C) employed a simple commercial at-
bed scanner as a detector for the colorimetric detection of protein
and DNA microarrays [5267]. Mirkin and coworkers developed a
scanometric DNA array system coupled with a signal enhance-
ment method based on the precipitation of silver on AuNP tags
[52]. The assay consisted of a captured DNA strand immobilized
on a glass chip that recognized the DNA of interest. Oligonucleo-
tide-functionalized nanoparticle probes and oligonucleotide
targets were then cohybridized to the substrate in buffer solution.
 Gold nanoparticle-based signal amplication / X. Cao et al. / Anal. Biochem. 417 (2011) 116
Fig.5. Schematic representation of the proposed CL immunoassay based on silver deposition on colloidal gold labels. GIgG, goat anti-human IgG; HIgG, human IgG. (From Ref.
[49].)
After catalytic reduction of silver onto the surfaces of AuNPs to am-
plify the signal, the captured strand/target/nanoparticle sandwich
was visualized with a atbed scanner (hence the term scanometric
is used to describe the approach). Target concentrations as low as
50 fM could be detected, which was a nearly 100-fold increase in
sensitivity over traditional uorescence-based assays. With similar
principles, AuNPs have been applied in ultrasensitive detection of
DNA hybridization, proteinprotein interactions, and carbohy-
drateprotein interactions [5356]. Xu and coworkers used hydro-
quinone (HQ) as the reduction agent to facilitate the AuNP-
catalyzed reduction of silver ions to amplify signals for the detec-
tion of cytokines. The assay used a sandwiched format with an
AuNP-modied aptamer and a biotin-modied aptamer to simul-
taneously bind the target protein. The absorbance signal generated
by the aptamerprotein complex was enhanced by the deposition
of silver on the surface of AuNPs [58]. The same amplication tech-
nique has been demonstrated in a convenient and label-free scano-
metric approach. This approach integrated the bioconjugation and
aggregation of glyconanoparticles, the specic recognition of lectin
by carbohydrate on the cell surface and glyconanoparticles, the
aggregation-regulated silver signal amplication, and the spot test
for in situ cell surface carbohydrate assay [61]. In this work, the
glyconanoparticle was obtained by conjugation of AuNPs with
mannan. These glyconanoparticles exhibited fast aggregation
when exposed to ConA. In the presence of mannose motif-abun-
dant cells, the lectin-induced aggregation could be inhibited by
specic binding of lectin to the cell surface carbohydrate to de-
crease the concentration of free lectin in the glyconanoparticle rec-
ognition system. The lectin-induced aggregation could be directly
and quickly visualized by an AuNP-catalyzed silver enhancement
process due to the fact that the aggregated AuNPs had lower silver
enhancement capability than dispersed AuNPs [62]. This provided
an easy-to-use method for in situ monitoring of cell surface
carbohydrates.
Ultrasensitive detection of carbohydrateprotein interaction
was achieved by magnetic preconcentration combined with silver
enhancement amplication technology [59]. The iron oxide/gold
core/shell nanoparticles were synthesized to visualize carbohy-
drateantibody or carbohydratelectin hybridization at very low
concentration. In this glycan array system, the iron core made it
possible for the magnetic eld to quickly bring nanoparticle-
5
labeled proteins or antibodies to the array of carbohydrates immo-
bilized on glass slides and to help them encounter the carbohy-
drates at very low concentration. The gold shell provided a
well-established platform for conjugation of biomolecules. When
coupled with a signal amplication method that was based on
nanoparticle-promoted reduction of silver, the sensitivity of an
iron oxide/gold core/shell nanoparticle-based assay reached the
subattomole level in carbohydrate detection. This result exceeded
that of the analogous uorophore system by 1 order of magnitude.
Surface-enhanced Raman scattering (SERS) is one of the most
sensitive diagnostic approaches available to the analytical chemist.
This approach is similar to the method that employed multiple
uorophores as labels; however, the spectroscopic lines in Raman
spectroscopy are not as broad as the bands in uorescence spec-
troscopy, and the spectral window is much broader. This, in princi-
ple, allows a greater degree of multiplexing. Furthermore, only
single-wavelength laser radiation is needed to scan a highly multi-
plex array with numerous target-specic Raman dyes. This is in
contrast to array-based detection of molecular uorophore probes,
where different excitation frequencies are needed for each uoro-
phore. Indeed, target DNA sequences specic to multiple different
bioterrorism agents have been identied using this approach with
spectroscopically distinguishable nanoparticle probes. Attachment
of Raman dye-labeled oligonucleotides to the AuNP probes gener-
ated spectroscopic codes for individual targets of interest, thereby
permitting multiplex detection of analytes [63,64]. Specically, the
presence of the target was conrmed by silver staining, and the
detection of the target was revealed by recording SERS signal of
the Raman dye near the nanoparticle surface (Fig. 6). The unoptim-
ized detection limit of this method was 20 fmol [63].
AuNP aggregates
The aggregation of AuNPs leads to the formation of a new
absorption band at longer wavelengths. This is a direct result of
electric dipoledipole interaction and coupling between the plas-
mons of neighboring particles in the formed aggregates. Oligonu-
cleotide bridged AuNP aggregates provoked a red-to-blue color
change [7678]. This simple phenomenon pointed out the possibil-
ity of AuNP as a DNA detection agent [11]. The milestone discovery
by Mirkin and Letsingers group [76] was that the color change
6 Gold nanoparticle-based signal amplication / X. Cao et al. / Anal. Biochem. 417 (2011) 116

Fig.6. Signal amplication with silver staining by using SERS approach. If Raman dyes (blue spheres) were attached to the labeling probe in the scanometric assay, the targets
could be encoded and detected via the Raman signal of their labels. (For interpretation of the reference to color in this gure legend, the reader is referred to the web version
of this article.) (From Ref. [63].)

Fig.7. Schematic for the amplied sandwich-type detection assay of the BRCA-1 gene using multifunctional crosslinked gold aggregates and magnetic particles. DNP,
detection nanoprobe; MMP, magnetic microparticles; CNP, crosslinking nanoprobe; TMB, 3,30 ,5,50 tetramethylbenzidine. (Note. The drawing is not to scale.) (From Ref. [82].)

resulting from AuNP aggregation could be applied for the quantita-
tive determination of DNA using colorimetric assay. Mercaptoalky-
loligonucleotide-modied AuNPs, averaging 13 nm in diameter,
were used as reporter groups. The hybridization of the probe and
the target sequence resulted not only in the binding of an oligonu-
cleotide probe to the target sequence but also in the formation of
an extended polymeric network where the reporter units were
interlocked by multiple short duplex segments. The signal for
hybridization was governed by the optical properties of the nano-
particles, which depended in part on their spacing within the poly-
meric aggregate. Although the detection system was not
optimized, it was able to detect 10 fmol of target DNA. Since then,
intensive DNA or DNA-related detection assays based on AuNP
aggregates have been developed [7781]. Zhao and coworkers re-
ported a sandwich-type assay for the optical detection of DNA
using multicomponent crosslinked AuNP aggregates (Fig. 7) [82].
The DNA-bridged gold aggregates, which were loaded with a high-
er amount of HRP, were employed as signal amplication labels. In
the presence of target DNA, the multicomponent detection nanop-
robes were attached to the magnetic microparticles (step I). The
complexes were further hybridized with nanoprobe to form a
crosslinked aggregate. Once magnetic eld was added, these sand-
wich complexes were magnetically separated (step II). As a result,
HRP conned at the surface of gold aggregates could catalyze the
enzyme substrate and generate an optical signal. While in the ab-
sence of target DNA, gold aggregates could not attach to magnetic
microparticles and, in turn, were not to be separated. The detection
limit was approximately 1 fmol.
Mirkins approach to DNA hybridization assays could be used to
quantify the level of antibodies in aqueous and serum samples on
 Gold nanoparticle-based signal amplication / X. Cao et al. / Anal. Biochem. 417 (2011) 116
the basis of the aggregation of AuNPs [16,83,84]. Thanh and Rosen-
zweig used AuNPs that averaged 10 nm in diameter, coated with
protein A, to determine the level of anti-protein A in aqueous
and serum solutions [16]. The aggregation of the AuNPs resulted
in an absorption change at 620 nm that was monitored by an
absorption plate reader. A dynamic range of 2 orders of magnitude,
along with a limit of detection of 1 lg ml1 of anti-protein A, was
observed. Zeng and coworkers described the phenomena that AuN-
Ps were brought together to form networks at the micrometer
scale after the addition of rabbit IgG, in which a large island-like
aggregation that consisted of hundreds of AuNPs was formed. As
is well known, each single-chain variable fragment (scFv) molecule
captured one arm of the rabbit IgG molecule and then shared the
antigen with a neighboring scFv molecule (two binding sites per
rabbit antibody). Consequently, when rabbit IgG was added into
the solution of scFv-stabilized AuNPs, aggregations occurred
(Fig. 8). The immunosensor showed high sensitivity with a detec-
tion limit of 1.7 nM of scFv [84].
Bio-barcode techniques
The bio-barcode assay [11,8590] is a promising new amplica-
tion and detection system that uses short oligonucleotides as tar-
get identication strands and surrogate amplication units in
both protein and nucleic acid detection. The typical assay involves
two types of particles. One is magnetic beads and has recognition
elements for the target of interest attached to their surface, and
the other is AuNPs functionalized with both target capture and
hundreds of barcode capture oligonucleotides that are hybridized
to barcode DNA. In the presence of target DNA, the magnetic
microparticles and the AuNPs form sandwich structures that are
magnetically separate from solution and washed with water to re-
move the hybridized barcode DNA. The barcodes are detected
using either a uorescence or scanometric method [90].
Most of the bio-barcode detection schemes still require micro-
arrayer-based immobilization of oligonucleotide on a glass chip,
surface passivation chemistry to minimize nonspecic binding, sil-
ver enhancement of immobilized AuNPs on a chip, light scattering
measurement, and a quantication step. Importantly, sophisticated
instruments such as microarrayers and chip imaging tools limit
portability, and the assay cost is high. To obviate or minimize the
above requirements without sacricing attomolar sensitivity of
the bio-barcode assay, Groves and coworkers reported an ultrasen-
sitive colorimetric bio-barcode amplication method that relied on
porous silica beads and an AuNP-based colorimetric DNA detection
scheme for straightforward readout [91]. The assay was based on
porous microparticles, which enabled the loading of a large
Fig.8. Scheme of the aggregation process of scFv-stabilized AuNPs after the addition of rabbit IgG. (From Ref. [84].)
7
number of barcode DNA per particle and AuNP-based colorimetric
barcode detection. During the detection procedure, only the simple
mixture and separation of probe solutions would result in attomo-
lar sensitivity without using a microarrayer and complicated signal
amplication steps such as enzymatic amplication and silver
enhancement or sophisticated signal measurement tools. The
detection limit was 30 aM concentration of cytokines, which was
approximately 3 orders of magnitude more sensitive than conven-
tional nonenzymatic cytokine detection assays and 15 times more
sensitive than an enzyme-based amplication method.
Zhang and coworkers reported a modied barcode amplica-
tion assay for ultrasensitive detection of Hantaan virus nucleocap-
sid protein (HNP) [92]. The modied assay was based on AuNP
enhancement combined with the immuno-PCR technique on an
ELISA plate. During the assay, the target antigen HNP was captured
by a polyclonal antibody coated on ELISA microplate wells, fol-
lowed by the addition of AuNPs, dually modied with oligonucle-
otides and an HNP-specic monoclonal antibody L13, to form a
sandwich immunocomplex. The oligonucleotides on the AuNP con-
tained two strands: one as captured DNA immobilized on the sur-
face of the AuNP through an AuS bond and the other as signal
amplication DNA that was partially complementary with the cap-
ture DNA. After the immunocomplex was formed, the signal DNA
was released by heat and was consequently characterized by
PCR/gel electrophoresis and SYBR Green real-time PCR. The detec-
tion limit of this method could be improved down to 10 fg ml1 for
detection of puried HNP in buffers as well as in human serum,
which was approximately 7 orders of magnitude more sensitive
than that of conventional ELISA.
Frster resonance energy transfer method
Frster resonance energy transfer (FRET), also called uores-
cence resonance energy transfer in some cases, is a mechanism that
describes energy transfer between two chromophores. An excited
state donor chromophore may transfer energy passed a certain dis-
tance (typically 38-nm range) to an acceptor chromophore
through nonradiative dipoledipole coupling. Muhammed et al.
showed an efcient FRET between the molecular gold cluster (a class
of compounds of gold called quantum clusters) Au25 and the dansyl
chromophore. Energy transfer from the dansyl donor to the Au25
core was manifested by way of the reduced lifetime of the excited
state of the former and drastic quenching of its uorescence. The
metal (especially AuNP)-enhanced FRET technique was widely used
for studies of the structural dynamics of biomolecules due to its high
sensitivity and simplicity for detection of ligand (receptor binding
observed merely by the enhanced signal of the acceptor) [93].
8 Gold nanoparticle-based signal amplication / X. Cao et al. / Anal. Biochem. 417 (2011) 116

Fig.9. Schematic illustration of the selection of quadruplex-binding ligand-based FRET assay using AuNPs as a uorescence quencher. GNP, gold nanoparticle. (From Ref.
[96].)

Fig.10. Schematic principle for assay of the glycoprotein based on the energy transfer between Lectin-modied QDs (Lec-QDs) and carbohydrate-conjugated AuNPs (Carbo-
AuNPs) on a glass slide. (A) strong PL signal from Lec-QDs immobilized on an amine-reactive glass surface (NHS-hydrogel slide); (B) low PL signal due to the energy transfer
between Lec-QDs (as a donor) and Carbo-AuNPs (as a quencher); (C) partial recovery of the PL in presence of glycoproteins because of the reduction in energy transfer
between Lec-QDs and Carbo-AuNPs due to competitive inhibition. (From Ref. [98].)

Ray et al. reviewed the development of the nanoparticle-based
FRET method for ssDNA sequence recognition and monitored the
cleavage of DNA by single-stranded-specic nucleases [94,95]. Jin
et al. designed a FRET system for selection of quadruplex-binding
ligands with unmodied AuNPs as a uorescence quencher. The as-
say was based on the properties of AuNP that ssDNA could be ab-
sorbed onto negatively charged AuNPs at a chosen ionic strength,
whereas the G-quadruplex does not at the same ionic strength
(Fig. 9). When a dye-labeled oligonucleotide single-stranded probe
was adhered to the AuNPs, the attendant proximity of the dye to
the gold led to uorescence quenching of the dye [96]. Zhang
and coworkers presented a simple recognition system with CdTe
QDs as donor and AuNPs as acceptor, linked with complementary
ssDNA to form CdTe-double-stranded DNAAuNP conjugates as
uorescence probe for DNA detection based on the FRET process
[97]. After the hybridization step, the FRET occurred because the
donors and acceptors were close enough to each other.
Kim et al. described a chip-based system for the detection of
protein glycosylation using FRET-based energy transfer between
QDs and AuNPs. The basic principle of this method relied on mod-
ulation of the energy transfer efciency between the lectin-modi-
ed QDs and carbohydrate-conjugated AuNPs by glycoproteins
[98]. Lectin-conjugated QDs (Lec-QDs) were immobilized on an
amine-reactive glass surface (NHShydrogel slide). The irradiation
of light onto the backside of the glass surface resulted in a strong
photoluminescence (PL) signal from the immobilized Lec-QDs
(Fig. 10A). When in close proximity to these luminescent QDs,
the carbohydrate-conjugated AuNPs (Carbo-AuNPs) functioned as
 Gold nanoparticle-based signal amplication / X. Cao et al. / Anal. Biochem. 417 (2011) 116
a quencher due to the binding afnity between the lectin and car-
bohydrate groups (Fig. 10B). However, if glycoproteins were also
present in the solution that contained the Carbo-AuNPs
(Fig. 10C), the glycan moiety on the glycoproteins would cause a
reduction in energy transfer between the Lec-QDs and Carbo-AuN-
Ps. This was due to competitive inhibition, which resulted in partial
recovery of the PL of the QDs.
Kim et al. developed a multiplexed assay system for proteases
along with the energy transfer between QDs and AuNPs on a glass
slide. In this system, the PL of donor QDs was immobilized on sur-
face and quenched due to the presence of acceptor AuNPs in close
proximity. Meanwhile, the protease activity caused modulations in
the efciency of energy transfer between the acceptor and donor
and, thus, enabled the protease assay. In comparison with the
QDdye system, the conjugates of the QDAuNP gave rise to higher
energy transfer efciency, which resulted in quantitative assay of
proteases with higher sensitivity [99].
Grant and coworkers designed a biosensor by a capture anti-
body labeled with a uorescent dye bound to a protein labeled
with an AuNP or a QD. Then FRET was performed with uorescent
dye as donor and with AuNPs or QDs as acceptor [100]. When ex-
posed to porcine reproductive and respiratory syndrome virus
(PRRSV), a binding event between the virus particle and antibody
took place, which resulted in a conformational change. This change
altered the distance between the uorescent dye and the nanopho-
tonic molecule, thereby altering the energy transfer that took place
between the uorescent dye and the QD or AuNP. Using this ap-
proach, they were able to rapidly and effectively detect PRRSV in
solution, with a detection limit of 3 particles ll1. Pihlasalo et al.
developed easy-to-use homogeneous method that used time-re-
solved uorescence resonance energy transfer (TR-FRET) and uo-
rescence quenching for quantication of eukaryotic cells [101]. The
method relied on a competitive adsorption of cells and uores-
cently labeled proteins onto citrate-stabilized colloidal AuNPs.
Fewer than ve cells were detected.
Song and Fans group combined the highly specic binding abil-
ities of aptamers with the ultra-high quenching ability of AuNPs to
develop a multicolor gold nanoprobe for the simultaneous detec-
tion of three analytes: adenosine, potassium ion, and cocaine. This
AuNP was fabricated by self-assembly of three dye-modied se-
quences on AuNPs. In their work, dyes were held in close proximity
to the AuNP surface through hybridization of assembled ssDNA
probes and dye-labeled aptamer sequences, which resulted in ef-
cient AuNP quenched uorescence. In the presence of targets, the
aptamertarget binding separated the duplex, liberated the dye-la-
beled aptamer into solution, and restored the uorescence. This
kind of three-color gold nanoprobe could simultaneously and
selectively detect adenosine, potassium ion, and cocaine with high
sensitivity [102].
Detection methods
Optical methods
AuNPs have been widely used in optical biosensing because of
their unique optical properties, especially plasma absorbance.
The spectral shifts that originated from adjacent or aggregated
AuNPs have attracted increased interest in the development of
optical biosensors based on biomaterialnanoparticle hybrid
systems.
Surface plasmon resonance
The bright colors of AuNPs, which is the basis for their applica-
tion as markers, are due to the presence of a plasmon absorption
band. This absorption band is produced when the incident photon
9
frequency is in resonance with the collective excitation of the con-
ductive electrons of the particle. This effect is termed localized sur-
face plasmon resonance (LSPR). AuNPs LSPRs usually occur
between 520 and 800 nm. Because of such special plasma absor-
bance, AuNPs have been extensively used in DNA [103] and anti-
bodyantigen [82,8486] analyses.
Cationic AuNPs were used for signal amplication through ionic
interaction with 16S ribosomal RNA (rRNA) hybridized on the pep-
tide nucleic acid probe-immobilized surface plasmon resonance
(SPR) sensor chip. 16S rRNA was used as a genetic marker for iden-
tication of organisms and could be analyzed directly without PCR
amplication due to the relatively high number of copies. The pep-
tide nucleic acid was a neutral backbone structure; therefore,
hybridization with 16S rRNA resulted in the ionic condition being
changed from neutral to negative. This method resulted in a detec-
tion limit of Escherichia coli rRNA of 58.21.37 pg ml1, which was
decreased approximately 5500-fold compared with that without
signal amplication. In addition, this method could be used for
Staphylococcus aureus detection without purication of rRNA
[104]. Oligonucleotide-capped AuNPs were used in a sandwich as-
say of oligonucleotide or polynucleotide by a ow injection SPR. A
carboxylated dextran lm was immobilized onto the SPR sensor
surface to eliminate nonspecic adsorption of oligonucleotide-
capped AuNPs. This also facilitated the signal enhancement associ-
ated with specic molecular recognition events. The signal
enhancement originated from the LSPR effect and the formation
of an additional layer of a high refractive index at the sensor sur-
face. A remarkable low DNA detection level could be achieved with
low sample consumption. For the analysis of 39-mer target DNA,
the detection limit was as low as 2.1  1020 mol, which corre-
sponded to 1.38 fM. The method was shown to be reproducible
(relative standard deviations [RSDs] < 16%) and possessed high se-
quence specicity [105].
Hsu and Huang developed a sensitive technique that used the
rolling circle amplication (RCA) postsignal processing method
for analyzing signals on protein microarray through the SPR meth-
od [106]. They chose oligonucleotide-modied AuNPs as the detec-
tion probe. Use of the AuNPs tags led to a greater increase in angle
shift, which allowed improvements in sensitivity compared with
other non-AuNP-assisted binding events. Huang et al. developed
an automated microuidic integrated LSPR-based biosensor in
which AuNPs were synthesized in solution and immobilized on
quartz substrates [107]. The developed microuidic integrated sys-
tem was capable of transporting a specic amount of biosamples
into the sensor chambers, which achieved sensitive and specic
biosensing with less reaction time and reagent consumption.
The spectral position of the LSPR band is strongly dependent on
the particle size and shape. The anisotropic gold nanostructures
LSPR peaks, such as gold nanorods and gold nanocages, could be
easily tuned by changes in the synthesis parameters. This allowed
the control of the aspect ratio (AR) and, therefore, showed greater
enhancement in biosensing as markers [108]. Drastic sensitivity
enhancement was obtained through longitudinal plasmonic reso-
nance of gold nanorods for ultrasensitive SPR biosensing. This pro-
cedure used functionalized gold nanorods as amplication labels
due to the electromagnetic interactions between the nanotag and
the sensing lm. The detection sensitivity of the nanorod-conju-
gated anti-rabbit antibody for rabbit IgG was estimated to be
40 pg ml1 [108].
Fluorescence detection
Fluorescence detection is critical in cell imaging, target recogni-
tion, disease diagnosis, and fundamental studies of biological pro-
cesses. Metallic nanoparticles have been shown to enhance the
uorescence emission and decrease the molecular excited state
lifetimes of vicinal uorophores [87,91,93]. Such an enhancement
10 Gold nanoparticle-based signal amplication / X. Cao et al. / Anal. Biochem. 417 (2011) 116
was a result of interactions between uorophores and surface plas-
mons in metallic nanostructures. Therefore, AuNPs have been used
to enhance the sensitivity of uorescence measurement with the
SPR method. A ber-optic biosensor, based on a localized surface
plasmon coupled uorescence (LSPCF) system, was developed
[109]. This LSPCF biosensor was able to detect mouse IgG at a min-
imum concentration of 1 pg ml1 (7 fM) during the biomolecular
interaction of the IgG with anti-mouse IgG. The results conrmed
very high sensitivity due to the amplication of LSPCF intensity
by AuNP coupling.
Fluorescent molecules emitted at wavelengths in the physiolog-
ically relevant water window (700900 nm) were of particular
interest. This was due to the large penetration depth of near infra-
red (NIR) light in most biological media and offered the potential
for imaging at signicant depths in living tissues. However, it has
been proven that it was rather difcult to achieve bright uores-
cent with photostable and biocompatible NIR uorophores. Aniso-
tropic gold nanostructures, such as gold nanorods and gold
nanoshells, have proven to be excellent materials due to longitudi-
nal plasmonic resonance, which could be tuned to the NIR wave-
lengths [110,111]. The uorescence enhancement of the NIR
uorophore IR800 by gold nanoshells and gold nanorods has been
recorded. Human serum albumin (HSA) served as a spacer layer be-
tween the nanoparticle and the uorophore. The measurements re-
vealed that the quantum yield of IR800 was enhanced from 7%, as
an isolated uorophore, to 86% in nanoshellHSAIR800 complex
and 74% in nanorodHSAIR800 complex. This dramatic increase
in uorescence showed tremendous potential for contrast
enhancement in uorescence-based bioimaging.
Scattering detection
On exciting the LSPR of AuNPs or a nanostructured substrate,
the large electromagnetic elds that localized at the surface of
the gold nanostructure can be exploited to measure enhanced
vibrational molecular signatures. This occurs through the phenom-
enon known as surface-enhanced Raman scattering. SERS is a pow-
erful spectroscopic technique that provides nondestructive and
ultrasensitive characterization down to the single molecular level,
comparable to single-molecule uorescence spectroscopy. It has
been used for sequence-specic DNA detection [63,112,113] and
protein detection [114]. The aggregation of AuNPs caused by the
specic recognition between the biomolecules carried by AuNPs
and target biomolecules or by deposition of silver on the surface
of AuNPs was responsible for the signal enhancement. Most inter-
esting and also important, the SERS could be turned on and off by
biomolecular binding and dissociation event. The mechanisms of
such controlled metal nanoparticle-based SERS for DNA recogni-
tion and signal amplication were rst demonstrated by Graham
et al. [115] and further conrmed by Nie and coworkers [112]. Dra-
matic changes occurred in both the optical absorption and surface-
enhanced Raman spectra of the 60-nm SERS beacons excited at
785 nm. This suggested that sequence-specic DNA hybridization
brought two or more particles into a distance range and, thus, re-
sulted in plasmonic coupling and further enhancement of the Ra-
man signals [112].
Dynamic light scattering (DLS), also known as photon correla-
tion spectroscopy or quasi-elastic light scattering, is a technique
routinely used to analyze the size and size distribution of poly-
mers, proteins, colloids, and nanoparticles. Because of the strong
light scattering property of AuNPs, it is natural to hypothesize that
DLS can be a very sensitive technique for quantitative detection
and analysis of nanoparticle probes at low concentration. A one-
step, wash-free, and amplication-free assay for prostate-specic
antigen (PSA) [116] analysis using AuNPs coupled with the DLS
technique was developed for the rst time in 2008 by Liu et al.
Anti-PSA-conjugated AuNPs (anti-PSA AuNPs) were mixed with a
sample solution that contained free PSA (f-PSA) antigen. The bind-
ing of f-PSA caused nanoparticles to form dimers, oligomers, or
aggregates, depending on the concentration of the antigen. A very
low concentration (0.5 ng ml1 for PSA detection) could be de-
tected though DLS analysis [116]. The capability of such a method
was later testied using mouse IgG as the target protein by the
same research group [117].
Resonance light scattering (RLS) techniques based on self-
assembly of AuNPs have been exploited for small molecule
[118,119], DNA [120], and protein [7376] detection. The large size
of the assemblies of AuNPs and the surface plasmon coupling
among AuNPs can enhance RLS signals. A nano-gold-labeled
immunoresonance scattering spectral method was established by
combining an immunoreaction and the resonance scattering ef-
fects of AuNPs [121]. This method showed high sensitivity and
good selectivity for quantitative determination of apolipoprotein
AI (ApoAI) and ApoB in the presence of polyethylene glycol (which
served as stabilizer to AuNPs) in human serum. Recently, RLS has
been carried out for the detection of adenosine in human urine.
The sensitive method was based on the specic recognition and
signal amplication of adenosine aptamer-modied AuNPs, which
was fabricated by triggering a structure switch of the 30-terminus
G-rich sequence and aptamer duplex via G-quartet-induced nano-
particle assembly. RLS signal linearly correlated with the concen-
tration of adenosine over the range of 6115 nM. The limit of
detection for adenosine was 1.8 nM with RSDs of 2.904.80%
(n = 6).
Table 1 lists some AuNP-based amplication optical detection
methods with details.
Electrochemical methods
Certainly, electrochemical techniques are the most widely
used methods in biomaterial analysis and sensing applications
based on metal and semiconductor nanoparticles. This is due to
their relatively low cost and the widespread diffusion of the re-
lated instruments required for such techniques in addition to en-
hanced sensitivities achieved by new electrode materials during
recent decades [41]. AuNPs and their various nanostructures have
been used to achieve direct wiring of enzymes to electrode sur-
face. This promoted electrochemical reaction to impose nano-bar-
code for biomaterials and to amplify signal of biorecognition
event [140].
Voltammetric methods
The application of voltammetric techniques for AuNP-based sig-
nal amplication for biosensing was based on two detection meth-
odologies. The rst one was direct methods that did not involve
any of the prechemical dissolution of AuNPs. The second was based
on previous dissolution of AuNPs and analysis of the correspondent
ions or catalytic methods by other species. Merkoi and coworkers
developed a geomagnetic sensor for detection of DNA hybridiza-
tion. They used magnetic graphiteepoxy composite electrodes to
accumulate gold tags. This involved the hybridization between a
capture DNA strand, which was linked with paramagnetic beads,
and another DNA strand related to the BRCA1 breast cancer gene,
which was coupled with streptavidin-coated AuNPs. The electro-
chemical detection of AuNPs by differential pulse voltammetry
(DPV) showed that the sensor provided reliable discrimination
against noncomplementary DNA as well as against one- and
three-base mismatches [141]. This detection methodology was
also illustrated by Kerman and coworkers [142]. They employed
a biotinylation of the kinase substrate and a biotin-modied aden-
osine 50 -triphosphate [c]-biotinyl-3,6,9-trioxaundecanediamine as
the cosubstrate. After the phosphorylation and biotinylation of the
peptides, the attachment of streptavidin-coated AuNPs onto the
 Gold nanoparticle-based signal amplication / X. Cao et al. / Anal. Biochem. 417 (2011) 116 11

Table 1
Some AuNP-based amplication optical detection methods.

Detection method AuNP Amplication times Target(s) Detection Linearity range(s) Reference
diameter limit(s)
(nm)
SPR detection
Surface-enhanced plasmon resonance 15 DNA [103]
SPR 30 10 times bare chip Salmonella 104105 cells ml1 [122]
typhimurium cells
SPR AChEACL1; ACL2 7 pM; 12 pM 0.0110 nM; 0.02 [123]
1.2 nM
LSPR-coupled uorescence 20 12-fold IgG 1 pg ml1, 7 fM 01 ng ml1 [109]
LSPR 13.5 BiotinBSA DNA [124]
1
SPR 25 12.5-fold Testosterone 3.7 pg ml [125]
Luminescence detection
ECL 16 Human IgG 1.68 ng ml1 5100 ng ml1 [19]
ECL 12 BSA: 10; IgG: 6 BSA; IgG 1 lg ml1; 10120 lg ml1; [20]
5 lg ml1 5100 lg ml1
ECL 12 QDs provide 1.7 order of Human prealbumin 1  1011 g ml1 5  1010 to [22]
magnitude on AuNPAu 1  106 g ml1
electrode
CL immunoassay AFP 0.01 ng ml1 0.15.0 ng ml1 [31]
ECL 20 Human IgG 1 pg ml1 7.5100 pg ml1 [39]
CL 16 Human IgG 5 pg ml1 0.0250 ng ml1 [49]
Photoelectrochemical 15 DNA 1 nM 1500 nM [126]
Light scattering detection
Optical signal 15 1000 Cytokine 83 aM 1 fM100 pM [58]
Light scattering spectra 10-fold DNA: BRCA-1 gene 1 fM [82]
SERS-plasmonic coupling 40/60 40200 DNA [112]
Enhanced RLS Urinary adenosine 1.8 nM 6115 nM [118]
Resonance Rayleigh scattering 20 1000 Transferrin 85 pM 85 pM3.4 nM [127]
50
Resonance scattering 9 ApoAI; ApoB 2.04 lg L1; 0.0083 0.333 mg L1; [121]
0.96 lg L1 0.00200.197 mg L1
Light scattering detection 13 1000-fold Human IgG 10 ng ml1 0.0510 lg ml1 [83]
Hyper-Rayleigh scattering 10 10 times colorimetric by 10 lg ml1 [128]
UVvis
DLS 40 [129]
SERS 46 4; 1010; 250-fold 1,5-Naphthalenedithiol zmol [130]
41 8
SERS Glucose [131]
Colorimetric/scanometric detection
Charge-coupled device image 8.512 Human IgG 0.0510 ng ml1 [57]
Scanometric image Carbohydrateprotein 100 fM 1091016 M [59]
interaction
Scanometric image 15; 20 1000 times ELISA PSA 1 fg ml1 104 range [89]
Scanometric image 5 IgG 1 ng ml1 [60]
Colorimetric immunoassay 15 IgG 1.7 nM 1.7170 nM [84]
Scanometric image Comparable to PCR HeLa cell 10 cells [132]
Scanometric 13 10,000 PSA, AFP, human 300 aM in buffer [133]
chorionic gonadotropin 2 fM in serum
Scanometric image 40 8 DNA 2 fmol 0500 fmol [134]
Camera image 10; 40 100 Troponin 0.01 ng ml1 0.1014.27 ng ml1 [135]
Colorimetry 13 10,000 Single-nucleotide 70 fM [136]
polymorphism
genotyping
ELISA 2 CA15-3 060 U ml1 [30]
Colorimetric image 30 107 times ELISA Hantaan virus 10 fg ml1 [92]
Colorimetric image >14 1000-fold DNA; DNA cleavage 0.5 fmol [137]
Colorimetric image 13.7 0.8 DNA 1 pmol [138]
Colorimetric image 9 Blood group [139]

Note: AChE, acetylcholinesterase, ALC1 and ALC2, AuNPs labeled carbamate inhibitors.

surface of surface plasmon-coupled emission via biotinstreptavi-
din afnity was performed. Direct DPV analysis of the captured
AuNPs enabled the monitoring of the activity of the kinase and
its substrate as well as the quantitative information about the ex-
tent of the phosphorylation and biotinylation reactions.
High sensitivity could be obtained when the indirect detection
methods were performed. As mentioned before, such methods
were either based on predissolution of AuNPs and analysis of the
corresponding ions [143,144] or based on catalytic deposition of
other species such as silver [42,44,47], copper [68,69], and p-nitro-
phenol [32] using gold as catalyst [145]. Much attention has been
given to the latter strategies, which yielded high sensitivities. The
enhancement in sensitivity for an electrochemical immunoassay,
by the catalytic deposition of Au3+ onto AuNPs in the presence of
reduction agent, has been studied [145]. The chemical deposition
of Au3+ on the AuNP-labeled proteins enlarged the square wave
voltammetry (SWV) signals. This allowed the author to sensitively
detect analyte with a detection limit of 1.6 fM protein. This limit
was 3 orders of magnitude lower than that obtained by a conven-
tional immunoassay that used the same AuNP label.
12 Gold nanoparticle-based signal amplication / X. Cao et al. / Anal. Biochem. 417 (2011) 116

Fig.11. Schematic representation of successive fabrication, human IgG detection (5 ng ml1), and regeneration of an MEA platform-based immunosensor. EDC/NHS, 1-ethyl-
3-(diethylaminopropyl)carbodiimide/N-hydroxysuccinimide. (From Ref. [148].)

Table 2
Some AuNP-based amplication electrochemical detection methods.

Detection method AuNP diameter (nm) Amplication times Target(s) Detection Linearity range(s) Reference
limit(s)
Voltammetric detection
ASV 20 4.7 IgG 1 ng ml1 1.6627.25 ng ml1 [44]
ASV 13 Human IgG 0.5 ng ml1 2250 ng ml1 [68]
ASV 15 Salmonella typhi 98.9 cfu ml1 1.30  102 to [69]
2.6  103 cfu ml1
ASV 16; 5 200 DNA 0.5 fM 1 pM1 nM [149]
AC voltammetry 13 300% IgG 10 pg ml1 251000 pg ml1 [24]
CV Comparable to the AFP 0.6 ng ml1 155 ng ml1 [23]
immunoradiometric assay
CV DNA 1 fM 1 fM0.1 lM [32]
CV 15 3 orders of magnitude Human IgG 23 fg ml1 5  108 to [47]
7.5  107 lg ml1
CV CA15-3 0.5 U ml1 2.5120 U ml1 [150]
CV 16 2.28 HBsAg 0.1 ng ml1 0.5650 ng ml1 [151]
CV AuNP seeds: 13; Au- 50 CEA; AFP 4 pg ml1; 0.0480 ng ml1; [152]
PB-Fe3O4: 80 7 pg ml1 0.074142 ng ml1
CV 10 Comparable to bio-barcode Protein, mouse IgG 1 fg ml1, 7 aM 1 fg ml1 to [153]
assay 10 lg ml1
CV AuNW (200 nm Label free without Testosterone 0.1 ng ml1 1.283.5 ng ml1 [154]
diameter, 10 lm length) mediator
1 1
CV 16 AFP 0.23 ng ml 1.25200 ng ml [155]
CV 16 CEA 0.06 ng ml1 0.2120 ng ml1 [156]
CV 10 Mouse IgG 1 fg ml1 (CV) 1 fg ml1 to [157]
100 lg ml1
CV 16 Human IgG 8  107 M 5.0  106 to [158]
9.6  104 M
DPV 13 AFP 1.0500 ng ml1; [27]
0.8 ng ml1
1
DPV 20 Human IgG 40 pg ml 0.12580 ng ml1 [33]
DPV 10 Mouse IgG 100 ag ml1 1 fg ml1 to [157]
100 lg ml1
DPV 20; 41; 71.5 HBsAg 2360 ng ml1 [159]
DPV 10 Thrombin 7.82 aM 74.7 aM1.5 fM [160]
Amperometric detection
it Plot 20200 3-fold Estradiol 6 pg ml1 1200 pg ml1 [161]
it Curve 5; 10; 15 Ascorbic acid, dopamine, and 1.5  107 M 5.1  107 to [162]
hydrogen peroxide (ascorbic acid) 6.7  104 M
Electrochemical impedance spectroscopy
EIS 5 Protein A 1 ng ml1 [50]
EIS 15 1000 times lower IgG 36 pg ml1 0.05100 ng ml1 [148]
(human IgG)
EIS 25 Salmonella spp. 102 cfu ml1 102105 cfu ml1 [163]
EIS 2.56 2 orders of magnitude ApoAI 50 pg ml1 0.110 ng ml1 [164]
EIS 10 Fluorescein 15 ng ml1 [165]

Note: CV, cyclic voltammetry; it, currenttime; PB, Prussian blue; AuNW, gold nano-wire; HbSAb, Hepatitis B surface antigen; CEA, Carcinoembryonic antigen; CA 15-3,
cancer 15-3; ApoAI, apolipoprotein A-I.

Electrochemical impedance spectroscopy resistance occurring at conductive or semiconductive surfaces.

Electrochemical impedance spectroscopy (EIS) is a very power- Electrochemical transformations that occurred at the electrode/
ful tool for the analysis of changes in interfacial capacitance or electrolyte interface can be modeled by extraction of components
 Gold nanoparticle-based signal amplication / X. Cao et al. / Anal. Biochem. 417 (2011) 116
in the electronic equivalent circuits that corresponded to the
experimental impedance spectra [146]. AuNPs have recently been
used in impedimetry-based biosensing applications with the main
goal of amplifying electrical signals generated by biorecognition
events that occurred at the electrode surface. Bonannia et al.
[147] recently used AuNPs for impedimetric signal amplication
in a DNA sensing device. In their work, the probe oligomer was ad-
sorbed onto a glassy carbon electrode surface and the impedance
measurement was performed in a solution containing the redox
marker ferrocyanide/ferricyanide. They used streptavidin-coated
AuNPs for specic binding of the target biotinylated oligomer after
the hybridization event. A silver enhancement treatment was per-
formed at the end to visualize the hybridization extension on the
electrode surface. This technique offered further signal amplica-
tion. Yu and coworkers introduced a renewable, site-selective
immobilization platform of microelectrode array (MEA) for multi-
plexed immunoassays by using pencil graphite particles coated
with gold layers as microelectrodes (Fig. 11) [148]. The results
showed that the developed MEA sensor allowed detection of hu-
man IgG with a wide linear range (0.05100 ng ml1) and more
than three times higher sensitivity than that of the conventional
gold electrode. The primary antibodies were site selective and
immobilized on this platform to selectively capture their antigens
from the sample media. The subsequent introduction of AuNP-la-
beled antibodies resulted in a signicant decrease in the interfacial
electron transfer resistance. In addition, this site-selective immobi-
lization platform could be easily regenerated by a simple electro-
chemical treatment.
Table 2 lists some AuNP-based amplication electrochemical
detection methods with details. Although the specic electrochem-
ical methods listed were used for quantitative measurement of the
substrates in each reference, other electrochemical or optical
methods were also employed for characterization of the sensing
interface/interphase conditions. This gave more information such
as the inuence of different nanoparticles size or type, electro-
chemical behaviors, and performances of the sensor.
Inductively coupled plasma mass spectrometry
Inductively coupled plasma mass spectrometry (ICP-MS) has
proved to be one of the most sensitive techniques for trace element
analysis, with upsides including large dynamic range, low detec-
tion limits, and multielement and rapid analysis capability [166].
By taking advantage of its high sensitivity and wide linear dynamic
range, ICP-MS was used as a powerful detection method in biosen-
sing with the signal amplications of AuNPs [166,167]. Recently
Zhao et al. demonstrated a novel sandwich assay for human a-
thrombin that used two afnity aptamers for increasing specicity,
AuNPs for signal amplication, and ICP-MS for highly sensitive
detection [168]. The rst aptamer, Apt29, was conjugated to AuNPs
(10 nm). The second aptamer, Apt15, was attached to streptavidin-
coated magnetic beads (1 lm). The binding of the two aptamers to
the same thrombin molecule resulted in a sandwich complex
(Apt29thrombinApt15). The use of magnetic beads facilitated
the capture of targets in homogeneous solution and provided fast
and efcient magnetic separation of the afnity complexes. While
the magnetic beads were removed, the AuNPs in the solution were
analyzed by ICP-MS. The detection limit of human a-thrombin was
as low as 0.5 fmol. The dynamic range covered approximately 3 or-
ders of magnitude.
An afnity assay for bacterial cells using AuNP labeling and ICP-
MS detection was described for the rst time by Li et al. [169]. The
target bacterial cells bound with the antibody to form afnity com-
plexes. This linked the cell, antibody, and AuNPs. The mixture was
centrifuged to separate the AuNPs bound to the cells from the
unbound AuNPs. The fraction of the cell-bound AuNPs was then
13
acid-digested and quantied, through the determination of gold,
using ICP-MS. A rapid and sensitive assay for the E. coli O157:H7
bacteria could be completed with antibody afnity binding, AuNP
labeling, and ICP-MS detection. The assay was able to detect as
few as 500 E. coli O157:H7 cells in 1 ml of sample (500 cfu ml1).
Tests with nonpathogenic E. coli (DH5a, ATCC35218, and
ATCC25922) showed high specicity of the assay for E. coli
O157:H7. Each assay was completed within 40 min. Demonstra-
tion of this assay for E. coli O157:H7 suggested potential for detec-
tion of a variety of bacterial pathogens.
Conclusion and outlook
The studies described above demonstrated the potential of bio-
conjugated nanoparticles for amplication transduction of biorec-
ognition events. Understanding of AuNPs nanostructures and their
role in the catalytic events was a critical step forward for biosen-
sing applications. Given the enormous amplication afforded by
AuNPs, remarkable sensitivity for detection of small molecules,
disease markers, biothreat agents, infectious agents, and cells
was achieved.
Successful realization of the new signal amplication strategies
required proper attention to nonspecic adsorption issues that
commonly controlled the detectability of bioafnity assays. Thus,
proper washing and surface blocking steps should be employed
to avoid amplication of background signals (associated with non-
specic adsorption of the nanoparticle ampliers). These strategies
included the use of AuNPs as carriers for both probe and signal
molecule loading, as indicators to induce the deposition of silver
on AuNPs for both optical and conductivity detection, as labels in
coupling with bio-barcode techniques, and as receptors when
employing FRET methods. Recent work, based on FRET for the
investigation of metal and semiconductor nanoparticles and QDs,
illustrated the extraordinary sensitivity achievable by this tech-
nique. In conclusion, the extraordinary physical and chemical
properties of structures available for AuNPs, along with their appli-
cations, will promote fundamental studies in connection with
those of other inorganic/organic molecules. This potential enables
biomaterials in multi-/interdisciplinary research involving various
sciences (e.g., physics, chemistry, biology, food, health care), agri-
culture, and industry.
Acknowledgments
The project was supported by the Key Program (21035002)
from the National Natural Science Foundation of China, the Na-
tional Natural Science Foundation of China (20875013), the Key
Program (BK2010059) from the Natural Science Foundation of
Jiangsu Province, the Technology Support Program of Jiangsu Prov-
ince (SBE200900289), and the Open Research Fund of State Key
Laboratory of Bioelectronics, Southeast University. We gratefully
acknowledge D. Shen (Canada) for polishing the English.
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