Trends in Analytical Chemistry 65 (2015) 83–96

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Trends in Analytical Chemistry

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a c

Colorimetric detection of mercury(II) based on gold nanoparticles,

fluorescent gold nanoclusters and other gold-based nanomaterials
Woravith Chansuvarn a,*, Thawatchai Tuntulani b, Apichat Imyim b,*
a Division of Science, Faculty of Science and Technology, Rajamangala University of Technology Phra Nakhon (RMUTP), Bangkok 10800, Thailand
b Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

A R T I C L E I N F O A B S T R A C T

Keywords:
This review focuses on colorimetric and visual assays for determination of mercury(II) (Hg2+) ions based
Colorimetry
on gold nanoparticles (AuNPs), fluorescent gold nanoclusters (AuNCs), gold nanorods (AuNRs), gold
Fluorescence
Gold nanocluster
nanoflowers (AuNFs) and gold nanostars (AuNSs). The analytical colorimetric approaches based on AuNPs
Gold nanoflower can be categorized according to the aggregation or disaggregation mechanisms of functionalized or un-
Gold nanoparticle modified AuNPs. The colorimetric concept involves change of the color of a solution of AuNPs from deep
Gold nanorod red to purple or blue. Disaggregation assays involve reversing the color from blue to red. Detection using
Gold nanostar AuNCs is based on Hg2+-induced fluorescence quenching of functionalized AuNCs and label-free AuNCs.
Mercury(II) The benefits of these systems are simplicity, rapidity, cost-effective production, ease of usage, easy vi-
Naked eye sualization of the color change, and detection using spectrophotometry, fluorometry and the naked eye.
Test strip
We summarize applications to real samples, limits of detection, and the development of test strips.
© 2014 Published by Elsevier B.V.

Contents

1. Introduction and background .......................................................................................................................................................................................................................... 84

2. Synthesis and functionalization of gold nanoparticles .......................................................................................................................................................................... 84
2.1. Synthesis of gold nanoparticles ........................................................................................................................................................................................................ 84
2.2. Functionalization of gold nanoparticles ........................................................................................................................................................................................ 84
3. Optical property of gold nanoparticles ........................................................................................................................................................................................................ 85
4. Mercury(II) detection with colorimetric assays using gold nanoparticles ...................................................................................................................................... 85
4.1. Colorimetric methods based on aggregation of functionalized gold nanoparticles ....................................................................................................... 85
4.1.1. DNA-functionalized gold nanoparticles ......................................................................................................................................................................... 85
4.1.2. Non-DNA functionalized gold nanoparticles ............................................................................................................................................................... 86
4.2. Colorimetric methods based on aggregation of unmodified gold nanoparticles and removal of protecting groups from the surface of gold
nanoparticles ........................................................................................................................................................................................................................................... 88
4.3. Colorimetric detection based on disaggregation of gold nanoparticles induced by certain ligands ........................................................................ 88
4.4. Colorimetric assays based on direct reduction of gold(III) to gold nanoparticles (in-situ assay) ............................................................................. 89
5. Mercury(II) detection base on fluorescent gold nanoclusters ............................................................................................................................................................. 90
5.1. Detection methods based on functionalized gold nanoclusters ............................................................................................................................................ 91
5.2. Detection methods based on label-free gold nanoclusters ..................................................................................................................................................... 92
6. Development of test strips ............................................................................................................................................................................................................................... 92
6.1. Test strips based on gold nanoparticles ......................................................................................................................................................................................... 92
6.2. Test strips based on gold nanoclusters .......................................................................................................................................................................................... 92
7. Other gold-based nanomaterials for mercury(II) detection ................................................................................................................................................................. 92
7.1. Detection methods based on gold nanorods ................................................................................................................................................................................ 92
7.2. Detection methods based on gold nanoflowers and gold nanostars ................................................................................................................................... 93
8. Conclusion and future trends .......................................................................................................................................................................................................................... 93
Acknowledgements ............................................................................................................................................................................................................................................. 94
References .............................................................................................................................................................................................................................................................. 94

* Corresponding author. Tel.: +66 2218 7607; fax: +66 2218 7607.
E-mail address: woravith.c@rmutp.ac.th (W.Chansuvarn); iapichat@chula.ac.th (A. Imyim).

http://dx.doi.org/10.1016/j.trac.2014.10.013
0165-9936/© 2014 Published by Elsevier B.V.
84 W. Chansuvarn et al./Trends in Analytical Chemistry 65 (2015) 83–96
1. Introduction and background
Among nanomaterials, gold nanoparticles (AuNPs) have re-
ceived great attention for various aspects, such as visual sensors,
detection schemes and imaging analysis. The visual detection of the
mercury(II) (Hg2+) ion is most extensive due to its serious toxicity
causing environmental and human health problems. One of the
unique optical properties of AuNPs is surface-plasmon resonance
(SPR) or localized SPR (LSPR), the coherent oscillation of the me-
tallic free electrons in resonance with the frequency of the
electromagnetic field. The Lorenz-Mie theory described the SPR effect
(so-called Mie scattering) of spherical AuNPs [1,2]. The SPR of AuNPs
has a strong absorption band in the visible region and results in the
origin of the observed color effect displaying a maximum absorp-
tion band at a wavelength of about 520 nm, so it exhibits a deep-
red color [3]. The colorimetric behavior of AuNPs is related to their
SPR, which strongly depends on particle size, shape, geometry, re-
fractive index of the medium, interparticle distance and aggregation
state in solution. Any change in these parameters changes the
plasmon-resonance frequency, affecting the color and the strength
of the plasmonic enhancement that affects intensity [4–6]. Based
on the particle-size dependence of optical properties, AuNPs of 13-
nm and 56-nm diameter exhibit large SPR-extinction bands centered
at wavelengths of 520 nm and 530 nm, and the solutions show their
visible color as rose-red and purple-red, respectively [7]. A solu-
tion of smaller individual AuNPs (5–20 nm) appears deep red, while
those of larger particles or aggregates of small particles appear purple
to deep blue. The maximum SPR-absorption bands of AuNPs in-
crease with increasing mean diameter of AuNPs [8,9].
In the past decade, colorimetric detection and visual sensing of
Hg2+ based on functionalized AuNPs have been widely reported in
many areas of research because AuNPs exhibit high molar extinc-
tion coefficients and optical properties greatly dependent on size
and distance. Color change is easily monitored by the naked eye
without resorting to any advanced instruments. Synthesis tech-
niques and functionalization steps of AuNPs have been developing
continually, leading to improvements in selectivity and sensitivity
for ultra-trace levels of Hg2+ in various samples.
The functionalization strategy involves AuNPs being modified by
ligands or receptor molecules on the surface of the AuNPs through
gold-sulfur (Au-S) and/or gold-nitrogen (Au-N) bonds. AuNPs without
ligand capping are called unmodified AuNPs. However, in both strat-
egies, the synthesis of AuNPs is required first.
Within colorimetric analysis, aggregation of AuNPs is the main
mechanism, leading to a change in SPR (color and absorbance or
extinction), so AuNPs are particularly interesting in many re-
search applications due to their tunable optical properties. The limit
of detection (LOD) of AuNP-based colorimetric assays without signal-
amplification steps is in the range nM to μM.
Gold nanoclusters (AuNCs) have become promising lumines-
cent nanomaterials for detection of Hg2+ due to high selectivity and
ultra-sensitivity. The LOD can be as low as 0.01 nM (0.002 ppb).
AuNCs comprise a group of several to tens of Au atoms. Their cluster
size is typically in the range <1–2 nm. They have molecule-like prop-
erties, such as luminescence and fluorescence. The optical properties
of AuNCs depend on their size and stabilizing molecules bound on
the surface. Their emission wavelengths are in the UV, visible to near-
infrared regions (385–866 nm). AuNCs can be prepared by several
methods (e.g., microwave-assisted, sonochemical, photoreductive,
etching-based, and protein-assisted synthesis) [10].
In this review, we discuss the synthesis and the optical proper-
ties of AuNPs by focusing on the colorimetric method and visual
assays for Hg2+ detection. The development of an analytical method
is to achieve not only the highest sensitivity and the greatest se-
lectivity, but also simplicity, cost effectiveness, rapidity and reliability.
We therefore review up-to-date methods and their applications for
visual sensing and/or colorimetric detection of Hg2+ ions using AuNPs
prepared in different ways, including:
(1) functionalized AuNPs;
(2) unmodified AuNPs;
(3) disaggregated AuNPs; and,
(4) formation of AuNPs using direct reduction and deposition of
Hg0 onto the surface of AuNPs.
In addition, we review and discuss the detection of Hg2+ based
on fluorescent AuNCs, gold nanorods (AuNRs), gold nanoflowers
(AuNFs) and gold nannostars (AuNSs).
2. Synthesis and functionalization of gold nanoparticles
2.1. Synthesis of gold nanoparticles
The synthesis of AuNPs was demonstrated in the two most com-
monly used methods, including chemical reduction and ligand
passivation (so-called Brust-Schiffrin method). Both methods are
based on the use of chemicals to reduce gold(III) salt to gold(0), yield-
ing AuNP colloids [4,11,12].The reductants act as not only reducing
agent, but also stabilizer to protect AuNPs from self-aggregation.
The chemical method is well known for using the reduction of
HAuCl4 with tri-sodium citrate in water at boiling point in a compli-
cated multi-step process [13], resulting in colloidal AuNPs (so-called
the citrate-capped AuNPs, or as-prepared or as-synthesized AuNPs);
it was introduced by Turkevich et al. in 1951 [14]. This method re-
quires a citrate ion as reducing agent and, at the same time, the surface
of the AuNPs is passivated via chemisorption and/or physisorption by
citrate molecules [5]. The size of such AuNPs is controlled by varying
the citrate/Au3+ molar ratio from 5.0 to 0.5, to obtain the core size of
10–150 nm [15]. Citrate species can stabilize AuNPs; however, they are
readily replaced by other desired ligands that form stronger interac-
tions with the AuNP surface in so-called functionalization assembly (e.g.,
thiols, DNA, oligonucleotides, and peptides) for further applications.
Similar to tri-sodium citrate, gallic acid [16,17] and glucose [18] can be
used as reducing and stabilizing agents in preparing AuNPs. Sodium
borohydride (NaBH4) is a more powerful reducing agent that can reduce
gold(III) ions at room temperature [19]. Lin et al. [20] demonstrated the
easy two-step functionalization of thiols on the surface of citrate-
capped AuNPs. The citrate adsorbed on the surface was first displaced
by the negative charge of thiolic acid with a strong interaction to the
surface of AuNPs, resulting in monolayer encapping via disulfide-
bonded AuNPs.
The other approach, the Brust-Schiffrin method, is a two-phase syn-
thesis that is the most widely used for thiol-stabilized AuNPs in organic
solvents. A solution of HAuCl4 was transferred into toluene contain-
ing a cationic surfactant, tetraoctylammonium bromide, and thiolalkanes
or aminoalkanes as the phase-transfer agent. The toluene solution was
thoroughly stirred with an aqueous solution of NaBH4, to obtain smaller
particle sizes (1–30 nm) and a narrow size distribution. The surface of
the AuNPs was shielded by specific functionalities of ligands by place-
exchange reactions. The protection with poly(benzyl ether) dendrons
was pioneered with particle size 3–25 nm [21]. Other reducing agents
were developed with the dual roles of effectively reducing potentials
and powerful stabilization and dispersion of AuNPs. Dumur et al.
[22] reviewed and discussed particle-size-controlled synthesis of seven
types of reducing and capping agents (e.g., microorganisms, bacteria,
plant extracts, organic molecules, acids and salts, liposomes, polymer
and inorganic reagents).
2.2. Functionalization of gold nanoparticles
The stability of AuNPs can be related to their ability to prevent
self-aggregation or degradation. The functionalization methods have
 W. Chansuvarn et al./Trends in Analytical Chemistry 65 (2015) 83–96
used chemical interactions between recognition molecules and the
AuNP surface. The dual roles of functionalized AuNPs are to pre-
serve stability, functionality and unique optical properties of AuNPs
(e.g., strong plasmon-absorption band and light scattering) and to
retain their specific recognition properties toward target analytes,
leading to increased selectivity and sensitivity.
Generally, citrate ions are only weakly bound to the surface of
AuNPs, so they can be easily replaced by substituting ligands through
chemical interactions between the affinitive ligands and the AuNP
surface [23,24]. Using this concept, many biological and non-
biological molecules have been constructed to cap the whole AuNP
surface and/or partial substituents on the AuNP surface through
physical interactions (electrostatic and hydrophobic), covalent cou-
pling bonds and specific recognition interactions [6,22]. The
recognition ligands containing at least a thiol group (mercapto)
[20,25–33] can be chemically modified on the AuNP surface through
strong bonding of gold-sulfur (Au-S) or gold-nitrogen (Au-N), while
their positive and/or negative charges can act as stabilizers by elec-
trostatic interactions.
3. Optical property of gold nanoparticles
The main advantage of colorimetric sensor based on AuNPs is
their extremely high molar extinction coefficient, ε (M-1 cm-1) which
is called molar absorptivity in classical UV-visible spectroscopy. The
ε of AuNPs in the visible region is higher than the magnitude of con-
ventional organic chromophores or dyes by about 3–5 orders [34].
For example, the molar extinction coefficients of 13-nm and 50-
nm diameter AuNPs are 2.7 × 108 M-1 cm-1 and 1.5 × 1010 M-1 cm-1 (at
~520 nm), respectively [35–37].
UV-vis absorption spectroscopy has been used to determine the
molar extinction coefficient of AuNPs with different sizes and dif-
ferent capping-ligand monolayers, which are imaged by means of
transmission electron microscopy (TEM). The molar extinction co-
efficient of AuNPs is determined according to the Beer-Lambert Law.
Jain et al. theoretically calculated the molar extinction coefficient
of 40-nm AuNPs to be 7.66 × 109 M-1 cm-1 at a plasmon-resonance-
wavelength maximum of 528 nm [38]. This value is higher than other
strongly absorbing dyes, such as an indocyanine green (ε = 1.08 × 104 M-
1 cm ), rhodamine-6G (ε = 1.16 × 10 M cm ) and malachite green
-1 5 -1 -1
(ε = 1.49 × 10 M cm ). This result is consistent with Liu et al. report-
5 -1 -1
ing the molar extinction coefficients of different core sizes (D) of citrate-
capped AuNPs (4.61 ± 0.48 nm, 8.55 ± 0.79 nm, 20.60 ± 1.62 nm,
25.67 ± 5.62 nm and 34.46 ± 4.34 nm) in the range of 106–109 M-
-1
1 cm [39]. The decrease in core diameter of AuNPs provided a dramatic,
continuous increase in the molar extinction coefficient [40].
4. Mercury(II) detection with colorimetric assays using
gold nanoparticles
Analyte-induced aggregation of AuNPs is the main sensing mech-
anism, leading to a change in color and absorbance. In addition, a
color change of colloidal AuNPs through analyte-induced disas-
sembly and deposition of analytes onto AuNP surfaces has been
employed in the determination of Hg2+ ions in various samples. More-
over, the sensitivity and the selectivity of AuNP sensing depend on
many factors, such as particle size, concentration of AuNPs, nature
and density of the recognition-target analytes, pH, ionic strength,
composition of the buffer solution, and temperature.
In the past decade, colorimetric assays based on AuNPs and their
aggregation have been widely reported in many research areas. Thus,
only colorimetric schemes were reviewed and summarized with four
strategies of AuNP assembly, including:
(i) functionalized AuNPs;
(ii) unmodified AuNPs;
85
(iii) disaggregation of AuNPs; and,
(iv) formation of AuNPs using direct reduction and direct
deposition of elemental mercury (Hg0) onto the surface of
AuNPs.
4.1. Colorimetric methods based on aggregation of functionalized
gold nanoparticles
4.1.1. DNA-functionalized gold nanoparticles
Several ways have been employed to attract biological mol-
ecules containing simultaneous acidic and basic groups, such as DNA,
oligonucleotides and proteins, for effectively functionalizing and sta-
bilizing AuNPs. Electrostatic interaction between AuNPs and various
biological molecules is one of the easiest ways to functionalize and
to stabilize AuNPs. The positive charges of AuNPs can bind to neg-
atively charged and nucleophilic moieties with stable ionic
interactions. Generally AuNPs functionalized with biological mol-
ecules occurs through passive adsorption of ligands on the AuNP
surface by electrostatic and hydrophobic interactions. For citrate-
capped AuNPs, a strong negative charge on the citrate-stabilized
AuNP surface provides an opportunity for Coulombic interactions
with amine (NH2) groups of biological ligands adsorbed on the
NP surface. Meanwhile, thiolated biomolecules can be directly
bound to the AuNP surface via thiol-gold (S-Au) affinity interac-
tions [41].
Based on the affinity between single-stranded DNA (ssDNA)
and the surface of AuNPs through electrostatic attractions, the
uncoiling conformation of oligonucleotides can stabilize colloidal
AuNPs against their aggregation under high ionic strength, result-
ing in the red color remaining in the solution [20,42,43]. It is well
known that the Hg2+ ion has highly affinitive binding with the
thymine-thymine (T-T) base-pair residues in the DNA structure
[44–47].
The order of binding ability of mercury species towards DNA is
MeHg+~PhHg+ > EtHg+ > Hg2+ [48]. In the presence of Hg2+, an ssDNA
conformation was induced by changing the sequence of base pairs
to double-strand DNA (dsDNA) or duplex forms via T-Hg2+-T coor-
dination [49–51]. The dsDNA can be desorbed from the surface of
AuNPs and/or also changed from linear to hairpin structure, result-
ing in destabilizing power, and leading to AuNP self-aggregation.
It is also known that base pairs of DNA are able to adsorb onto
AuNP surfaces via ion-dipole interactions. Thus, a variety of
colorimetric sensors based on thymine-containing DNA/
DNAzyme functionalized-AuNPs has been widely employed
for selective detection of Hg 2+ in aqueous media based on
thiolate-DNA functionalized-AuNPs and the T-T mismatch concept
[52–54].
AuNPs functionalized with a different DNA sequence of thymi-
dine moieties was exploited for effective selectivity toward Hg2+ over
other competing ions. In this system, the melting temperature (Tm)
of hybridized DNA-AuNPs proportionally correlates with the con-
centration of Hg2+ [47,55,56]. Liu et al. designed poly Tn-ssDNA
adsorbed on the AuNP surface through electrostatic attraction for
the detection of Hg2+ at a nanomolar level. The detection mecha-
nism was represented by the formation of DNA-Hg2+complex through
T-Hg2+-T coordination, resulting in the conformation of poly Tn-
ssDNA change to folded structure [57]. By comparing with a number
of thymine base pairs (n), the stability of Tn-ssDNA-AuNPs in-
creased when n decreased, and the T33-AuNP assay provided the
highest sensitivity toward Hg2+ ions.
The colorimetric sensitivity of poly-Tn-adsorbed AuNPs toward
Hg2+ through Hg2+-induced aggregation can be accelerated with about
100-fold improvement upon adding 0.2 mM Mn2+, while, without
Mn2+, it was not improved by changes in the length of the T-base
in DNA or by increasing the incubation time [58].
86 W. Chansuvarn et al./Trends in Analytical Chemistry 65 (2015) 83–96

Fig. 1. Cysteine-modified gold nanoparticles (Cys-AuNPs). {Reprinted with permission from [28], ©2010 IOP Publishing}.

Similarly, different nucleotides were also constructed as specif- the kinetics of aggregated AuNPs was driven slowly by the
ically colorimetric probes toward Hg2+with an LOD of 0.05–0.3 μM interparticle-crosslinking process of analyte-induced aggregation
[59–61]. (Fig. 1) [28]. The selectivity of this assay could be pointed out with
the stability constants (log Kf) of metallic ions with cysteine; 43.5,
4.1.2. Non-DNA functionalized gold nanoparticles 16, 18, 17, 19, 12 and 4 for Hg2+, Co2+, Zn2+, Cd2+, Ni2+, Pb2+ and Mg2+,
It is well known that thiol sub-unit biomolecules can be direct- respectively [30]. The lowest detectable concentration of Hg2+ by
ly attached on the surface of AuNPs through Au-S affinity the naked eye was 15 μM [30]. With the assistance of UV radia-
interactions. Kim et al. discovered 11-mercaptoundecanoic acid tion to improve sensitivity, the detectable minimum concentration
(MUA)-capped AuNPs with a high molar extinction coefficient of was lowered to 100 nM. The same aggregation protocol of inter-
6 × 10-8 M-1 cm-1 at 526 nm [37]. The aggregation mechanism, re- particle crosslinking, dithioerythritol-modified AuNPs (DTET-
lating to color change (rose-red to blue) is driven by Hg2+ chelation, AuNPs) was demonstrated to bind Hg2+ through specific S-Hg2+-S
leading to the closer distance of AuNPs and broadening of the SPR interactions [29], as shown in Fig. 2. To improve the selectivity toward
band. the Hg2+ ion, EDTA was added to remove cationic interfering ions,
Similarly, 3-mercaptopropionic acid-functionalized AuNPs (MPA- such as Pb2+, Cd2+ and Cu2+. Similarly, Chen et al. [33] synthesized
AuNPs) were employed with 2,6-pyridinedicarboxylic acid (PDCA) N-1-(2-mercaptoethyl)thymine-functionalized AuNPs (T-S-AuNPs)
[7]. The role of Tris-borate-NaOH buffer solution (pH 9.0) and PDCA for Hg2+-ion sensing with an LOD as low as 2.8 nM.
is imperative for stabilizing MPA-AuNPs and improving selectivity Chemodosimeter functionalized-AuNPs were also synthesized
and sensitivity because they control surface density and metal che- from highly recognition ligands containing thiourea and sulfur moiety
lation. The sensitivity was improved by fine-tuning the buffer [65]. The detection mechanism employed was desulfurization in the
composition and the monolayer structure, achieving sensitivity less presence of Hg2+ [66,67]. Alkylaminopyrazole-modified AuNPs had
than 100 nM (20 ppb) of Hg2+ ions. Adenosine monophosphate (AMP)
was additionally modified on the MPA-AuNP system as MPA/AMP-
AuNP [25]. The MPA/AMP-AuNP solution can disperse in high-salt
concentration due to the high negative-charge density of AMP that
capped the surface of the AuNPs, while MPA-AuNPs can be aggre-
gated. Following the addition of Hg2+ions, coordination between the
carboxylic sub-unit of MPA and Hg2+allows the aggregation of AuNPs
via bridging of neighboring NPs. The selectivity was reported by the
stability constants between Hg2+ ions and chelating ligand MPA as
log K(Hg) = 10.1 [62]. To improve the selectivity toward Hg2+ ions
over competing metal ions, the PDCA ligand can bind to MPA-
AuNPs through Au-N bond with stability constants of metal ions with
PDCA as follows: log K(Hg) = 20.2, log K(Pb) = 8.2, log K(Cd) = 10.0
and log K(Mn) = 8.5 [63]. The sensitivity was enhanced by 10-fold
upon modifying Rhodamine6G to MPA/AMP-AuNPs. To enhance sen-
sitivity as low as 25 nM (5 ppb) Hg 2+ ions, MPA-HCys-PDCA-
AuNPs was employed in detection using a hyper-Rayleigh scattering
(HRS) technique [26]. To further improve the sensitivity with the
LOD of 10 nM (2.0 ppb), the MPA-HCys-AuNPs protocol in the pres-
ence of PDCA coupled with cloud-point extraction (CPE) was
introduced based on removing large AuNPs that could be ex-
tracted into a Triton X-114-rich phase for a few minutes, until the
surfactant-rich phase became nearly colorless. Unlike usual
colorimetric-based AuNPs with color change from red to blue, this
scheme is based on the color change from colorless to red due to
Hg2+ being associated with AuNPs transferring to the Triton X-114-
rich phase [64].
Similarly, L-cysteine can be used to stabilize AuNPs under high Fig. 2. Dithioerythritol-modified gold nanoparticles (DTET-AuNPs). {Reprinted with
salt conditions. With the concentration of Hg2+ions less than 20 μM, permission from [29], ©2010 American Chemical Society}.
 W. Chansuvarn et al./Trends in Analytical Chemistry 65 (2015) 83–96 87

a triple role in inducing, stabilizing AuNPs and using affinity to co-

Fig. 3. Aggregation mechanism based on direct deposit protocols of (a) Tween 20 stabilized-gold nanoparticles (AuNPs) {Reprinted with permission from [72], ©2010 American Chemical Society}, and (b) Tween 20-AuNPs
ordinate with metal ions [68].
According to non-crosslinking aggregation [5], diethylene,
triethylene and tetraethylene glycol thiols (HS-EG2, HS-EG3 and HS-
EG4, respectively)-capped-AuNPs [Au-S-EGn(n = 2, 3 and 4)] displayed
different aspects of colorimetric sensing toward Hg2+ detection [27].
Only Au-S-EG3 can show the obvious color change from red to blue,
while a couple of diethylene and tetraethylene glycols remained red
in color.
In this system, Hg2+ played an important role in breaking the Au-S
bonds prior to the aggregation process due to the extremely high
affinity of Hg2+ toward thiolate ligands. Liu et al. reported quater-
nary ammonium-capped-AuNPs (QA-AuNPs) as a selective Hg2+-
ion sensor without the addition of any other masking agents [30].
Hydrophilic (11-mercapto-undecyl)-trimethylammonium (MTA) con-
taining a quaternary ammonium-terminated thiol group was capped
on the surface of AuNPs via Au-S bonds. The aggregation of QA-
AuNPs immediately appeared after the addition of Hg2+ in acidic
solution because Hg2+ can abstract thiolates chemisorbed on the
AuNP surface, leading to the aggregation of AuNPs and change of
the color from red to blue within 1 min. The lowest detectable con-
centration of Hg2+ by the naked eye was 1 μM. With the assistance
of solar light irradiation for 30 s to improve the sensitivity, the lowest
detectable amount of this assay was lowered to 30 nM.
Similarly, nitrotriazole (NTA)-capped-AuNPs via nitrogen atoms
in the triazole ring showed colloidal stability after inducing aggre-
gation by Tris-buffer. NTA was also abstracted from the surface of
AuNPs because NTA can coordinate with Hg2+, leading to the ag-

incorporating ascorbic acid and N-acetyl-L-cysteine. {Reprinted with permission from [73], ©2011 American Chemical Society}.
gregation of AuNPs. This system provided an LOD as low as 7 nM
and 50 nM for spectrometry and visual detection, respectively [69].
Riboflavin (vitamin B2 form) was recently designed by an easy
surface modification, and it can protect AuNPs against Tris-buffer-
induced aggregation. It can be released from the AuNP surface by
forming the riboflavin-Hg2+ complex and leading to self-aggregation.
The method enables an excellent selectivity to detect Hg2+ over the
concentration range 0.02–0.80 μM, with an LOD of 14 nM [70]. Since
Hg2+ has high affinity with cyanuric acid (CA), a spectrometric ap-
proach based on CA-AuNPs was exploited in the presence of Tris-
HCl buffer (pH 7.4) and NaCl, remaining a red color. The stability
of CA-AuNP interparticle repulsion was reduced when Hg2+ formed
a CA-Hg2+-CA complex, leading to aggregation of AuNPs [71].
The novel aggregate mechanism based on the deposition of Hg0
onto the surface of AuNPs was discovered using the assembly of
Tween 20-stabilized AuNPs [72]. In this system, Tween 20 can ef-
fectively stabilize the citrate-capped AuNPs, prohibiting aggregation
of AuNPs. Hg2+ was reduced by excess citrate ions capped on the
surface of the AuNPs. The colorimetric detection was achieved by
surface deposition of mercury to form mercury-gold alloys. Tween
20 molecules were also desorbed from the AuNP surface, leading
to the aggregation of AuNPs, as shown in Fig. 3(a). To reduce the
incubation time and to increase the sensitivity, this system was de-
veloped by adding ascorbic acid in order to increase the reducing
potential. As demonstrated in Fig. 3(b), in the presence of Hg2+, it
would be reduced by using ascorbic acid to form an Hg-Au alloy
coating on the AuNP surface. This can inhibit the self-aggregation
of AuNPs induced by N-acetyl-L-cysteine [73].
Recently, Bi et al. [74] developed Tween 20-AuNPs in three forms
(nanorods, nanospheres and nanoplates). In this system, NaBH4 was
used instead of ascorbic acid for a powerful reduction reaction. Hg2+
was reduced to Hg0 by NaBH4 and then bound on the surface of
AuNPs to form a solid amalgam-like structure, which etched the
surface of the AuNPs and caused the SPR band of the AuNPs to
undergo a blue-shift and to decrease in intensity.
Recently, AuNPs functionalized by the dithiocarbamate deriva-
tive of calixarene were developed to detect Hg2+ in drinking-water
samples [75]. The sharp color change with red-shift and the color
88 W. Chansuvarn et al./Trends in Analytical Chemistry 65 (2015) 83–96

Table 1
Comparison of visual and colorimetric methods based on aggregation of functionalized gold nanoparticles (AuNPs) for detection of mercury(II) (Hg2+)

Surface-modified- Masking LOD (nM) LDR (μM) Time (min) Buffer/pH Sample Ref.
AuNP protocols agent

DNA-AuNPs PDCA 10 - 35 HEPES pH 7.4 - [51]

DNA-AuNPs - 60 0.1–10 10 HEPES pH 7.4 Fish tissue [54]
DNA-AuNPs - 100 0–2 10 - - [55]
DNA-AuNPs - 10 0–1 10 MOPs pH 7.5 Lake and drinking [56]
water
Poly-Tn-ssDNA-AuNPs PDCA 250 0.5–5.0 20 Na3PO4 pH 7.4 - [57]
T33-AuNPs/ Mn2+ PDCA 10 10–250 nM 6 PBS Pond water [58]
DNA-AuNPs PDCA 300 0.3–10 60 MSO - [59]
dTTPs-AuNPs - 50 0.2–0.6 10 Tris-HCl pH 7.2 Tab and river water [60]
Apt-AuNPs - 1000 - 5 - - [76]
DNA-AuNPs - 2.88 μM 0–60 - - - [77]
MPA-AuNPs PDCA 100 0.20–0.5 60 Tris-borate pH 9 - [7]
MPA/AMP-AuNPs - 500 0.5–3.5 30 PBS pH 7.4 - [25]
MPA-HCys-AuNPs PDCA 25 - 6–7 - - [26]
MPA-HCys-AuNPs - 10 10–500 nM 30 TX-114 Drinking water [64]
Cys-AuNPs - 100 10–200 nM 4–6 - - [28]
DTET-AuNPs EDTA 100 0.1–0.6 10 Na3PO4 pH 6.6 - [29]
Thymine-AuNPs - 2.8 0.005–1 5 pH 7.4 Tap water [33]
CHD-AuNPs - - 0–3.4 10 - - [65]
AAP-AuNPs - 50 0.05–0.5 - pH 3.2 - [68]
EGn-AuNPs - - - 60 HEPES pH 7.2 - [27]
QA-AuNPs 30 1000 30 nM-10 mM 1 pH 1 Drinking water [30]
NTA-AuNPs PDCA 1 0.01–0.5 - Tris pH 8 Lake water [69]
Riboflavin-AuNPs - 14 0.02–0.8 30 Tris-HCl pH 8.17 Synthetic [70]
CA-AuNPs - - 1.6–16 5 Tris-HCl pH7.4 - [71]
Peptide-AuNPs - 26 - 1 - - [78]
Peptide-AuNPs EDTA 20 μM 10–45 10 - - [79]
Acid protien-AuNPs - 1 μM 1 μM-50 mM - - - [80]
Tween 20-AuNPs EDTA 100 0.2–0.6 5 Na3PO4 pH7.9 Drinking and sea water [72]
Tween 20-AuNPs/AA EDTA 5 0.5–10 33 PBS Drinking and tap water [73]
Tween 20-AuNPs - - 5–100 nM 90 BR pH 10 Tap, lake and river water [74]

change from red to violet could be detected by the naked eye. The
LOD was 200 nM (40 ppb). Dynamic light scattering (DLS) study and
TEM imaging suggested an average hydrodynamic diameter of
functionalized AuNPs. The mechanism of Hg2+ sensing was based
on the assembly of AuNPs in solution. Table 1 summarizes the colo-
rimetric assays based on functionalized AuNPs.
4.2. Colorimetric methods based on aggregation of unmodified gold
nanoparticles and removal of protecting groups from the surface of
gold nanoparticles
The protocol of unmodified AuNP-based colorimetric and visual
sensors involves use of as-prepared AuNPs and removal of protect-
ing groups from functionalized AuNPs. The surfaces of AuNPs are
therefore uncapped with ligand molecules or free from any capping
agents. This strategy also relies on the target-triggered, interpar-
ticle distance and size-dependent coloration similar to functionalized
AuNPs, but its aggregation is induced by removing protecting
molecules from AuNP surfaces rather than by crosslinking. Based
on the high affinity between Hg2+ ions and thymine through stable
T-Hg2+-T coordination, DNA oligonucleotides with T-T mismatches
retain a random coil form that can effectively protect AuNPs from
aggregation in the absence of Hg2+, but they can form duplex in the
presence of Hg2+ due to the fact that Hg2+ has effectively coordi-
nated and causing the non-complementary mismatch to fully
hybridize [Fig. 4(a)] [81]. AuNPs can interact differently with a short
ssDNA, with spontaneous adsorption on their surface with high af-
finity, resulting in the ssDNA-stabilized-AuNPs remaining in
dispersion and red in color upon adding salt [Fig. 4(b)] [81–83]. The
T-rich single-stranded oligonucleotides (5′-TTTTTTTTTT-3′) were
folded as a double-strand DNA (dsDNA) structures and stripped the
oligonucleotides away from the surface of AuNPs when they formed
the T-Hg2+-T complex [84,85]. According to this concept, the LOD
for Hg2+ was as low as 0.25 nM.
Wang et al. reported a dual detection of Hg2+ based on T-T mis-
matches and unmodified AuNPs [86]. T-rich oligonucleotides (5’-
GGTTGGTGTGGTTGG-3’) were conjugated with a fluorescent
molecule onto the end of the oligonucleotides in order to protect
aggregation of AuNPs. Upon the addition of Hg2+ and salt, the
complexation of Hg2+ with T-T mismatch sites formed a hairpin struc-
ture, causing the removal of oligonucleotides from the AuNP surface.
A one-step colorimetric method based on a thin layer of HgS
quantum dots (QDs) formed in situ on the surface of AuNPs was re-
cently exploited for the high sensitivity of ultra-trace Hg2+ ions with
LODs of 0.486 nM and 5.0 μM for UV-vis and naked-eye methods,
respectively [87].
Dihydrogen sulfide (H2S) hydrolyzed from thioacetamide in acidic
condition reacted with the AuNP surface to form an Au-SH2 complex.
The HgS-QD was formed on the surface of AuNPs when Hg2+ reacted
with the Au-SH2 complex. Alternatively, Hg2+ was chemically reduced
using sodium citrate, and Hg0 adsorbed on the surface of AuNPs
through the amalgamation process [88].
Table 2 summarizes colorimetric assays based on unmodified-AuNPs.
4.3. Colorimetric detection based on disaggregation of gold
nanoparticles induced by certain ligands
In the past few years, the colorimetric approaches for Hg2+
detection based on the anti-aggregation mechanism of AuNPs
were exploited by observing the color-reverse-progression change
from blue to red. By taking advantage of the strong affinity of Hg2+
with certain ligands, many approaches were demonstrated for in-
hibiting aggregation of AuNPs through the ligand-induced
mechanism.
Yang et al. demonstrated an Hg2+-inhibited aggregation mech-
anism by pyridine-induced aggregation of AuNPs [90]. The dipole
interaction of the nitrogen atom of pyridine and Hg2+ is stronger
than that of the nitrogen atom and the AuNPs. The aggregation rate
 W. Chansuvarn et al./Trends in Analytical Chemistry 65 (2015) 83–96 89

Fig. 4. Unmodified protocols for gold nanoparticles (AuNPs) with (a) mercury-specific oligonucleotides {Reprinted with kind permission from [81], ©2008 Springer Science+Business
Media}, and (b) T-rich single-stranded oligonucleotides (5′-TTTTTTTTTT-3′) aptamer. {Reprinted with kind permission from [83], ©2009 Springer Science+Business Media}.

of 24-nm-diameter AuNPs was higher than that of 13 nm-diame-
ter AuNPs.
4-Mercaptobutanol (4-MB) can induce the aggregation of AuNPs
[91], whereas Hg2+ tends to form complexes with 4-MB through the
Hg2+-S bond with a higher stability constant than 4-MB with AuNPs
[92]. The aggregation mechanism showed that Hg2+ plays an im-
portant role in breaking Au-S bonds [91]. The LOD and the linear
dynamic range (LDR) were 500 nM and 1.0–7.5 μM, respectively.
Similarly, thymine molecules can induce aggregation of AuNPs
via Au-N dipole interaction, but can preferentially interact with Hg2+
to form a T-Hg complex through N-Hg bonds, such as T-Hg-T, re-
sulting in a red solution [93]. A linear correlation was obtained over
the range 2–12 μM with an LOD as low as 2 nM.
Based on the disaggregation concept, poly(diallyldimethylam-
monium) (PDDA) was used as a reducing agent for AuNP stabili-
zation [94]. Cysteine was used to associate with the surface of
positively-charged AuNPs through the Au-S bond and cross-
linking AuNPs via electrostatic interactions, resulting in the color
change from red to purple. Apparently, the color of colloidal AuNPs
became red upon addition of Hg2+ ions because of the formation
of the Hg2+-cysteine complex, which has a high affinity through the
Hg-S bond. This can then deactivate the aggregation. The influ-
ence of competing ions, especially Cu2+ and Pb2+ was also limited
using EDTA as masking agent.
Similarly, Hg2+ can disaggregate the AuNPs stabilized by 4,4’-
dipyridyl [95]. The principle of disaggregation of AuNPs after being
induced by 4-mercaptophenylboronic acid (MPBA) was reported for
the detection of Hg2+ [96]. The recognition mechanism is based on
the self-dehydration condensation of MPBA, resulting in the change
of color from blue to red. The LOD was 8 nM using UV-vis spectro-
photometry and was lower than the WHO standard.
Table 3 summarizes colorimetric methods based on disaggre-
gation of AuNPs induced by certain ligands.
4.4. Colorimetric assays based on direct reduction of gold(III) to gold
nanoparticles (in-situ assay)
A novel strategy was recently discovered based on a direct
formation of AuNPs via a simple reduction reaction. The direct
formation of AuNPs was first reported using NH 2 OH·HCl as a
mild reducing agent in the presence of Tween 20 stabilizer [97].
Without Hg 2+ , the color of the solution turned slowly to red
after the AuNPs formed. The speed of color change increased ob-
viously with increasing Hg2+ concentration. However, the reaction

Table 2
Comparison of visual and colorimetric methods for mercury(II) (Hg2+) detection based on aggregation of unmodified gold nanoparticles (AuNPs) and removal of protecting
groups from the surface of AuNPs

Ligand stabilizing/AuNPs LOD (nM) LDR (μM) Time (min) Buffer/pH Real sample Ref.

DNA/AuNPs 500 0–5 30 HEPES pH 7.4 - [82]

DNA/AuNPs 0.6 1.0 nM-0.1 mM 10 Tris-HCl pH 8 Tap and lake water [83]
DNA/AuNPs probe 250 0.75–1.5 20 Tris-OAc pH 7.4 Artificial water [85]
TBA/AuNPs 200 0.39–8.89 10 Tris-HAc pH 7.2 Lake water [86]
H2S/CTAB/AuNPs 0.485 0.01–80 90 pH 3.57 Pond water [87]
DNA/AuNPs 40 0.096–6.4 30 Tris-HCl pH7.4 Tap and river water [89]

Table 3
Comparison of visual and colorimetric methods for mercury(II) (Hg2+) detection based on disaggregation of gold nanoparticles (AuNPs) induced by certain ligands

Ligands LOD (nM) LDR (μM) Time (min) Buffer/pH Real sample Ref.

4-MB 500 1.0–7.5 150 glycine-NaOH pH 10.0 River water and soil [91]
PDDA-Cysteine 25 0.05–10.0 - HAc-NaAc pH 5.0 Laboratory water [94]
4,4’-Dipyridyl 15 0.04–1000 30 Tris-HCl pH 7.0 Tap and spring water [95]
MPBA 8 0.01–5.0 20 Citric-citrate pH 4.0 Lake and tap water [96]
90 W. Chansuvarn et al./Trends in Analytical Chemistry 65 (2015) 83–96
0.50
0.40
Absorbance
0.30
0.20
0.10
0.00
400
3-AEPE-AuNPs
Fig. 5. Mechanism and TEM images of the direct reduction of gold(III) to gold nanoparticles stabilized by 2-[3-(2-amino-ethylsulfanyl)-propylsulfanyl]-ethylamine (3-AEPE).
(Adapted with kind permission from [98], ©Springer Science+Business Media}.
lasted for 20 min and only 30–40% of Hg2+ was reduced and en-
capsulated inside or bound outside the AuNPs formed. This system
was also applied to determine Hg2+ in lake water with an LOD of
10 nM.
To accelerate the reaction rate further, Chansuvarn and Imyim
recently demonstrated rapid, simple reduction of Au3+ ions (in HAuCl4
form) with NaBH4 in the presence of a dithia-diaza ligand (2-[3-
(2-amino-ethylsulfanyl)-propylsulfanyl]-ethylamine, 3-AEPE) and a
co-stabilizer surfactant (Triton X-100) [98]. A 3-AEPE ligand con-
taining two sulfur (2S) and two nitrogen (2N) atoms as electron-
donor atoms was used as a stabilizer to prevent aggregation of AuNPs
and to provide high selectivity to Hg2+ ions, according to the HSAB
principle [hard and soft (Lewis) acids and bases]. The mechanism
of 3-AEPE-stabilised AuNPs was also described by coordination
bonding through the Au-S and Au-N, similar to the strong –SH
binding on the surface of AuNPs [99]. The solution of 3-AEPE-
stabilized AuNPs displayed a rose-red color, while its color changed
to blue rapidly within a few seconds of adding borohydride in the
presence of Hg2+ (Fig. 5).
This method offers many advantages, such as simplicity, rapid-
ity, and cost effectiveness, and does not require sophisticated
instruments. It provides a possible method for monitoring Hg2+ in
real water samples. Moreover, this method has several potential
advantages as an optical sensor, especially as synthesis of as-
prepared AuNPs is not required and observation time is shorter.
Table 4
Comparison of colorimetric methods for mercury(II) (Hg2+) detection based on direct reduction of gold(III) [Au(III)] to gold nanoparticles (AuNPs)
Reducing agent/stabilizer LOD LDR (μM)
(nM)
NH2OH·HCl/Tween 20 10 1 nM-1 μM
NaBH4/AEPE-Triton X-100 35 1.5–9.0
500 600 700 800
Wavelength (nm)
3-AEPE-AuNPs-Hg
Table 4 compares the methods for Hg2+ detection based on direct
reduction of Au(III) to AuNPs.
5. Mercury(II) detection base on fluorescent gold nanoclusters
Several synthesis methods for AuNCs, such as microwave-
assisted, sonochemical, photoreductive, etching-based, and protein-
assisted synthesis, were reviewed by Cui et al. [10]. In order to control
the shape and the size of AuNCs, templated agents, such as pro-
teins and polymers, must be used in all methods of synthesis.
Microwave-assisted techniques involve microwave irradiation
applied to a mixture solution of Au(III) and a protein-templated agent
acting as a reducing agent and/or stabilizer.
The sonochemical method uses high-intensity ultrasound to
induce a chemical reaction, the formation and the growth of AuNCs.
This method offers uniform shapes, narrow size distribution and
high-purity AuNCs.
The photoreductive method is based on ultraviolet light irradi-
ating Au(III) precursors to form AuNCs without using reducing agents.
The etching-based strategy has been used to synthesize AuNCs
in the presence of excess thiols. Ligand-protected AuNPs are etched
by an excess of thiol molecules to form AuNCs.
Lastly, protein-assisted synthesis methods refer to the use of
ssDNA and proteins, such as bovine serum albumin (BSA), human
serum albumin (HSA), lysozyme and insulin, acting as a biotemplate
Time (min) Buffer/pH Real sample Ref.
20 Mops buffer/pH 7 Lake water [97]
few seconds pH 1.4 Drinking water [98]
 W. Chansuvarn et al./Trends in Analytical Chemistry 65 (2015) 83–96 91

Fig. 6. (a) Visual detection of mercury ions based on fluorescence resonance-energy transfer (FRET) between cationic-oligofluorene-substituted polyhedral oligomeric silsesquioxane
(POSSFF) and gold nanoclusters (AuNCs), and (b) chemical structure of POSSFF. {Reprinted with permission from [103], ©2011 American Chemical Society}.

for the preparation of AuNCs. The synthesis method is very simple
and can be performed in “one-pot” fashion under mild conditions.
The shape and the size distribution of AuNCs can be easily controlled.
The detection principle is based on quenching Hg2+-induced flu-
orescence. The fluorescent intensity of AuNCs (IFo) decreased upon
increasing the concentration of Hg2+. A Stern-Volmer plot (IFo/IF)
against [Hg2+] or a linear plot of fluorescence ratios [(IFo-IF)/IFo] at
certain wavelengths versus log [Hg2+] or a plot of relative fluores-
cence (IF/IFo) of AuNCs as a function of [Hg2+] is usually used as a
calibration curve. Similar to AuNPs, AuNCs need one or more sta-
bilizers or protecting molecules to avoid their self-aggregation. Some
researchers have tried to prepare un-protected AuNCs (called label-
free AuNCs) and applied for Hg2+ detection.
5.1. Detection methods based on functionalized gold nanoclusters
Huang et al. [100] reported the synthesis of highly fluorescent
11-mercaptoundecanoic acid (MUA)-protected AuNPs for nano-
sensing of Hg 2+ . The linear range of 0–10 4 nM from a plot of
fluorescence ratios at 520 nm versus log [Hg2+] was obtained. The
method exhibited an LOD of 5 nM with remarkable selectivity over
other metal ions.
Lin et al. [101] synthesized protein-modified AuNCs using lyso-
zyme type VI (Lys VI) and used them for the detection of Hg2+ and
CH3Hg+ based on Hg2+-induced fluorescence quenching. The LODs
were lowered to 3 pM and 4 nM for Hg2+ and CH3Hg+, respectively.
Trypsin-stabilized fluorescent AuNCs of average size 1 nm with
a red emission at 645 nm were synthesized and used for Hg2+ de-
tection in tap, mineral, and river water in Japan [102]. The LOD was
50 ± 10 nM (10 ± 2 ppb). The linear range was 50–600 nM (10–
120 ppb). The functionalized AuNCs were photochemically stable
and showed a highly selectivity over interfering metal ions (i.e. Cu2+,
Ni2+, Ca2+, Mg2+, Na+, Pb2+, Zn2+, Co2+, and Cd2+).
Pu et al. [103] developed a cationic-oligofluorene-substituted
polyhedral oligomeric silsesquioxane (POSSFF)/BSA-encapsulated-
AuNC hybrid complex for visual detection of Hg2+ (Fig. 6). The pink
fluorescence of the POSSFF/AuNC complex (emission at 665 nm)
turned blue (emission at 435 nm) in the presence of Hg2+ via flu-
orescence resonance-energy transfer (FRET). A plot of fluorescence
intensity change (ΔI) versus Hg2+ concentration was linear in the range
0–1.8 μM. A very low LOD of 0.1 nM (0.02 ppb) was achieved.
Dihydrolipoic acid (DHLA)-capped fluorescent AuNCs were syn-
thesized and tested for Hg2+ detection [104]. A plot of IF/IFo against
Hg2+ concentration was linear in the range 0.001–10 μM. The LOD
was 0.5 nM (0.1 ppb).
Chen et al. [105] synthesized BSA-encapped AuNCs for the se-
lective detection of Hg2+ ions in pond-water samples. The linearity
of IF/IFo at 660 nm against Hg2+ concentration was over the range
10–250 nM. The LOD was 4 nM (0.8 ppb).
One-pot synthesis of lysozyme type VI (Lys VI)-stabilized
AuNCs and Ag-AuNCs, including small and large AuNCs, for Hg2+
sensing was presented by Chen et al. [106]. Linear plots of the
92 W. Chansuvarn et al./Trends in Analytical Chemistry 65 (2015) 83–96

Table 5
Comparison of detection methods of mercury(II) (Hg2+) based on fluorescent gold nanocl;usters (AuNCs)

Ligand stabilizing/AuNCs LOD (nM) LDR (nM) Time (min) Buffer/pH Real sample Ref.

11-MUA 5 0-104 10 Tetraborate pH 9.2 Pond water [100]

Lys VI 3 pM 10 pM-1200 10 Phosphate pH 7.0 Seawater [101]
Trypsin 50 ± 10 50–600 - pH 8 - [102]
POSSFF/BSA 0.1 0–1.8 - Phosphate pH 7.0 - [103]
DHLA 0.5 1 nM-10 μM - Phosphate Living cells (imaging) [104]
BSA 0.8 10–250 40 Phosphate pH 7.0 Pond water [105]
Lys VI 1 6–100 15 - Tap water [106]
BSA-AuNCs-CdTe 9 131–710 2–3 ns Phosphate - [107]
BSA-AuNCs-EDTA 8 25–5000 - Phosphate pH 7.4 Tap, wastewater [108]
Au+(label-free) 0.5 1–20 - HEPES - [109]

fluorescence-intensity ratio (I613 nm/I471 nm) versus the concentra-
tion of Hg2+ were obtained. The method was applied to tap-water
analysis. The LOD was estimated to be 1 nM (0.2 ppb).
A nanocomposite of BSA-conjugated AuNCs and tripeptide-
capped CdTe-QDs was developed for detection of Hg2+ and F− ions
based on turn-off and turn-on photoluminescence (PL), respective-
ly [107]. In the presence of Hg2+, the PL of the composite was
quenched by 74%. A plot of I/I0 as a function of Hg2+ concentration
was linear in the range of 0.131–0.710 μM. The LOD was 9 nM (1.8
ppb). The quenching of the fluorescence of BSA-stabilized AuNCs
by Hg2+ and Cu2+ could occur, resulting in non-selectivity for Hg2+
sensing. The selectivity towards Hg2+ ions using BSA-AuNCs was im-
proved by adding EDTA as a masking agent for coexisting Cu2+ ions
[108]. The LOD was 8 nM (1.6 ppb).
5.2. Detection methods based on label-free gold nanoclusters
Xie et al. [109] developed a label-free method for the detection
of Hg2+ ions with high selectivity and sensitivity using AuNCs. A small
amount of Au+ was used to stabilize AuNCs prepared using a protein-
templated method. Hg2+ was reduced by NaBH4 to Hg0, which binds
more weakly with Au+, resulting in a lower fluorescence-quenching
efficiency on the AuNCs. The LOD was 0.5 nM (0.1 ppb), which was
more sensitive (three-fold) than those of DNA-functionalized AuNP
methods.
Recently, Park et al. [110] showed that Hg2+-induced quench-
ing of fluorescent AuNCs could be used to detect biological thiols
in blood serum. The detection mechanism of the thiols was based
on blocking the Hg2+ quenching of fluorescent AuNCs. The turn-on
sensor was selective for cysteine, glutathione and homocystein.
Table 5 summarizes the detection methods base on fluorescent
AuNCs.
6. Development of test strips
Easy-to-use, portable test strips present several advantages
over the methods employed in aqueous media. Botasini et al. [111]
summarized a few studies based on the immobilization of AuNPs
on a solid support and colorimetric test strips.
6.2. Test strips based on gold nanoclusters
Xie et al. [109] developed a test strip based on label-free AuNCs.
AuNCs were dispersed on a nitrocellulose membrane and subse-
quently encapsulated by BSA scaffolds. The background color of
nitrocellulose membrane under UV light was light green. When
dipped in Hg2+-ion solution, the color of the nitrocellulose strips
changed from green to purple. The concentrations of Hg2+ could be
determined by the naked eye.
A test strip based on AuNCs-loaded BSA/poly(ethylene oxide)
electrospun membrane (BSA/PEO-AuNCs) was introduced in 2013
by Cai et al. [115]. The as-prepared BSA/PEO-AuNCs could emit red
fluorescence under the excitation of visible light, and the fluores-
cence quenching by Hg2+ could be observed by the naked eye under
UV-lamp illumination (Fig. 7). Using a fluorometer, the linear re-
sponse to Hg2+ concentrations was in the range 0.5–75 nM (0.1–15
ppb) with an LOD of 57 pM (0.01 ppb).
7. Other gold-based nanomaterials for mercury(II) detection
Other gold-based nanomaterials, such as gold nanorods (AuNRs),
gold nanoflowers (AuNFs) and gold nanostars (AuNSs), have been
employed for sensing Hg2+. We summarize below detection methods
of AuNRs and AuNSs.
7.1. Detection methods based on gold nanorods
AuNRs possess two absorption bands (i.e., the longitudinal band
at > 600 nm and the transverse band around 520 nm when exposed
to incident light, due to two directional electron oscillations) [38].
The ratio of the plasmon bands (longitudinal/transverse) can change
as a function of the aspect ratio (length/breadth) of AuNRs. AuNRs
can also exhibit NIR absorption and scattering. These properties make
AuNRs good optical sensing materials for Hg2+ [116].
Wang et al. [117] reported the approach of end-to-end assem-
bly of AuNRs in the presence of thymine-rich DNA (T-T) by means

6.1. Test strips based on gold nanoparticles

The entire development of test strips was based on DNA-

functionalized AuNPs and nitrocellulose membrane was used as a
support pad [112–114].
The test-strip probe was used for on-site detection with LOD
lower than 3 nM (0.6 ppb) [112,113].
Fig. 7. Photograph of fibrous membrane of gold nanoclusters (AuNCs) loaded with
The signal-amplified lateral flow strip (SA-LFS) presented lower bovine serum albumin/poly(ethylene oxide) (BSA/PEO-AuNCs) (left) in the pres-
LODs of 0.005 ppb and 0.0015 ppb by the naked eye and UV-vis anal- ence of 5 μM mercury(II) (Hg2+) (right) under UV lamp (365 nm). {Reprinted with
ysis, respectively [114]. permission from [115], ©2013 Elsevier B.V.}.
 W. Chansuvarn et al./Trends in Analytical Chemistry 65 (2015) 83–96
of the specific formation of T-Hg2+-T complexes. The concentra-
tion range of Hg2+ was 1–15 μM (0.2–3 ppm).
Huang et al. [118] prepared AuNRs using a seed-mediated growth
method in the presence of cetyltrimethylammonium bromide (CTAB).
The AuNRs were assembled through Na3PO4 induction and used for
the ultra-sensitive detection of Hg2+ based on the change of LSPR
signals due to the amalgamation of Hg and Au. The LOD was
10-5 nM (0.00002 ppb).
Bi et al. [74] synthesized AuNRs, AuNPs and silver nanoplates
(AgNPTs), and used them for Hg2+ detection. The highest sensitiv-
ity for Hg2+ quantitation was obtained with AuNRs. Detection was
based on the amalgamation of Au and Hg(0), which was a product
of NaBH4 reduction. A linear relationship was obtained between the
wavelength shift of the longitudinal band of AuNRs and Hg2+ con-
centration with the lowest Hg2+ concentration of 10 nM (2 ppb). Tap-
and river-waters samples were analyzed by this approach. In the
same year, this research group proposed simple spectrophotomet-
ric determination of Hg2+ based on inhibiting the aggregation of
AuNRs by the stable complex of Hg2+-dithiothreitol [119]. The
ΔA780/A662 ratio increased linearly with the increase of Hg2+ con-
centration. The LOD was improved to 0.42 nM (0.084 ppb).
AuNR-based Hg2+ sensors used functionalized glass substrates
[120,121]. Chemnasiri and Hernandez [120] immobilized AuNRs on
aminopropyl-functionalized glass slides by dipping them in the so-
lution of AuNRs. The Au NR/glass slides were placed in the solution
of NaBH4 and Hg2+ was then added. Upon the addition of Hg2+, the
color of the Au NR/glass slides changed from red to blue and the
UV-vis absorbance measurements were performed. The linear cor-
relation between the wavelength shift of the longitudinal SPR band
(565–530 nm) and Hg2+ concentration was obtained. The LOD was
1.0 ppb.
Heider et al. [121] used (3-mercaptopropyl)trimethoxysilane to
modify glass substrates, which chemically bound AuNRs to produce
a portable, sensitive Hg2+ sensor based on NaBH4-reduced Hg(0). The
wavelength shift against Hg2+ concentration/nanorod was plotted.
The LOD was calculated to be 2.28 × 10-19 M Hg2+/nanorod.
Placido et al. [122] proposed Hg2+ ion-directed assembly of
AuNRs for Hg2+ detection. AuNRs were functionalized with an
N-alkylaminopyrazole ligand, 1-[2-(octylamino)ethyl]-3,5-
diphenylpyrazole (PyL). The PyL-AuNRs were assembled in the
presence of Hg2+, resulting in a change in the longitudinal SPR band
of the PyL-AuNRs. Hg2+ detection relied on the plot of wavelength
shift versus Hg2+ concentration. The LOD was as low as 3 ppt.
Measurement of circular dichroism (CD) was employed for de-
tection of Hg2+ based on the Hg2+-mediated T-T base pair of DNA
[123]. The CD-signal intensity of the DNA-functionalized AuNR-
ladder superstructures increased when the Hg2+ concentration was
increased. This method had an LOD of 0.03 ng/mL and the Hg2+ con-
centration range was 0.05–10 ng/mL.
Although unmodified and functionalized AuNRs show a remark-
able sensitivity towards Hg2+ detection, their studies remain difficult,
mainly due to the complicated methods of synthesis, and the further
functionalization to control their shape and their size. Hence, the
application of AuNRs to Hg2+ sensing is still limited.
7.2. Detection methods based on gold nanoflowers and
gold nanostars
Gold nanoflowers (AuNFs) or gold multi-spiked NPs were also
prepared and applied to Hg2+ detection. Nalawade et al. [124] in-
troduced the preparation method of AuNFs by adding NaOH acting
as an accelerator for the reduction of AuCl4- in ethylene glycol to
AuNFs and using polyvinylpyrrolidone (PVP) as a stabilizer. The mean
diameter of AuNFs was 75 ± 10 nm (Fig. 8). The authors reported
using AuNFs for the colorimetric detection of Hg2+ and Pb2+ ions in
real water samples [125]. The colorimetric sensing mechanism was
93
Fig. 8. Transmission electron microscopy (TEM) image of gold nanoflowers. {Re-
printed with permission from [124], ©2012 Elsevier B.V.}.
similar to that of AuNRs, based on changes in the absorption spectra
of AuNFs upon adding metal ions. Unlike AuNR-based Hg2+ detec-
tion, the selectivity of AuNFs towards metal ions was low. They
exhibited a wavelength shift in the presence of both Hg2+ and
Pb2+ ions.
Gold nanostars (AuNSs) were recently introduced for ultra-
sensitive detection of Hg 2+ using a surface-enhanced Raman
scattering (SERS) technique by Ma et al. [126]. The detection prin-
ciple involved a self-assembled AuNS dimer based on the Hg2+-
mediated T-T base pair of ssDNA. In the presence of Hg2+, the SERS
signal depended on the degree of self-assembled AuNS dimers with
which the electromagnetic field was greatly enhanced. The Raman
reporter molecule (4-aminothiophenol, 4-ATP) was used to measure
SERS intensity. The plot of Raman intensity 1083 cm-1 against Hg2+
concentrations was linear within the range 0.002–1 μg/L. The LOD
was lowered to 0.8 ng/L.
8. Conclusion and future trends
We focused on visual and colorimetric detection of Hg2+ based
on AuNPs and detection of Hg2+ based on fluorescent AuNCs. We
reviewed many approaches in the synthesis of AuNPs via a redox
reaction using several types of reducing agents and stabilization
methods of the prepared AuNPs in aqueous solution as well as
various detection mechanisms of Hg2+. Most of the recent works
focused on using thiols, DNA, oligonucleotides, peptides and other
biomolecules as the stabilizer for preventing the aggregation of
AuNPs. Although these bio-chemicals are commercially available,
their cost is relatively high compared to classical small molecules
that are simple to prepare and to employ, such as 4-mercaptobutanol
and a dithia-diaza ligand.
We believe that development of the synthesis and the evalua-
tion of these kinds of molecules for Hg2+ colorimetric determination
using an unsophisticated, cost-effective instrument (e.g., UV-
visible spectrophotometer) would be beneficial for the budget-
limited laboratory, the on-site detection and the real-time monitoring
of Hg2+ in the environment. In addition, AuNCs have become prom-
ising luminescent nanomaterials for detection of Hg2+ due to high
selectivity and ultra-sensitivity.
One of the drawbacks of AuNCs is that they require protein or
peptide molecules to protect self-aggregation. Only a few studies
focusing on fluorescent AuNCs have been published, especially the
preparation of un-protected AuNCs (called label-free AuNCs) and
94 W. Chansuvarn et al./Trends in Analytical Chemistry 65 (2015) 83–96
applications to Hg2+ detection. We also discussed applications of
AuNRs, AuNFs and AuNSs to the detection of Hg2+. We found only
a few research works. The main limitation of employing AuNRs,
AuNFs and AuNSs is the difficulty of synthesizing and functionalizing
them and low selectivity of AuNFs. Thus, there are still great op-
portunities to develop and to improve the sensitivity and the
selectivity of gold nanomaterials towards Hg2+.
Several types of real samples had been analyzed based on AuNPs,
such as fish tissue, natural surface water, drinking water and sea-
water. Overall LODs were in the range 0.5– ~ 3000 nM with a
response time of 1–90 min. However, most of the samples were
water, especially tap and drinking waters.
The extension of applications to other types of matrix-rich
samples, such as food, food additives, wastewater, sediment, soil,
and biological samples, will be a matter of interest. Also, fabrica-
tion of gold-based nanomaterials loaded to solid supports (e.g., paper,
membrane or glass slides) to give a more convenient, portable strip
test would benefit users more.
Acknowledgements
The authors thank the Faculty of Science and Technology,
Rajamangala University of Technology Phra Nakhon (RMUTP) and
the 90th Anniversary of Chulalongkorn University Fund.
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