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A Rapid Colorimetric Detection of Melamine in Raw Milk by Unmodified Gold

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Article in Food Analytical Methods · October 2013

DOI: 10.1007/s12161-013-9562-3

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Food Anal. Methods (2013) 6:1441–1447
DOI 10.1007/s12161-013-9562-3
A Rapid Colorimetric Detection of Melamine
in Raw Milk by Unmodified Gold Nanoparticles
Hai-bo Xing & Yuan-gen Wu & Shen-shan Zhan &
Pei Zhou
Received: 13 September 2012 / Accepted: 1 January 2013 / Published online: 13 March 2013
# Springer Science+Business Media New York 2013
Abstract We report a rapid, visual, facile colorimetric de-
tection of melamine in raw milk by unmodified gold nano-
particles (AuNPs). AuNPs can be induced by melamine from
dispersion to aggregation rapidly, along with the color change
from red to blue (or purple). After the optimization of reaction
conditions, the whole analysis only needs about 20 min includ-
ing the simple pretreatment we designed. The analysis can be
observed by the naked eye and simple to operate without
sophisticated instruments. In the raw milk, the present limit
of detection for melamine is down to ∼70 ppb. Furthermore,
we explore the interaction principle between melamine and
citrate-stabilized AuNPs in this work and establish the foun-
dation for modifying the AuNPs and optimizing the system of
AuNPs–melamine in the future.
Keywords Melamine . Colorimetric detection . Raw milk .
Unmodified gold nanoparticles . Interaction principle
H.-b. Xing : Y.-g. Wu : S.-s. Zhan : P. Zhou
School of Environmental Science and Engineering, Shanghai Jiao
Tong University, Shanghai, China
H.-b. Xing
e-mail: michaelyanzi@163.com
H.-b. Xing : Y.-g. Wu : S.-s. Zhan : P. Zhou
Key Laboratory of Urban Agriculture (South),
Ministry of Agriculture, Shanghai, China
P. Zhou (*)
School of Agriculture and Biology, Shanghai Jiao Tong University,
Shanghai, 200240, China
e-mail: peizhousjtu@163.com
P. Zhou
Bor S. Lug Food Safety Research Center, Shanghai Jiao Tong
University, Shanghai, China
H.-b. Xing : P. Zhou
The Center for Food Nutrition and Safety, Shanghai Jiao Tong
University, Shanghai, China
Introduction
Melamine (1,3,5-triazine-2,4,6-triamine) is a industrial chem-
ical compound which is widely used in plastics, fertilizer,
melamino-formaldehyde resin, paper, and other products
(Yang et al. 2009). For its high nitrogen content (66 % by
mass), it is illegality added into food, feed, and even milk in
order to increase the apparent protein content based on the total
nitrogen, which is measured by the conventional standard
Kjeldahl and Dumas tests (Serdiuk et al. 2010). Recently,
melamine is considered to have low toxicity and the LD50 of
rat given with oral administration is greater than 3 g/kg. Small
amounts of occasional intake are nontoxic for the human body,
but long ingestion of melamine may cause reproductive and
urinary system damage, leading to bladder and kidney stones
and inducing bladder cancer (Cao et al. 2009). In 2008, in
China, high levels of melamine were found in milk and other
dairy products, causing kidney failure and stones in ten
thousands of infants. In 2007, in USA, protein feeds for pets
mixed with melamine killed lots of cats and dogs (Choi et al.
2010). Therefore, in 2008, The World Health Organization's
food safety experts decided that the amount of melamine a
person could stand per day, the “tolerable daily intake,” was
0.2 mg/kg of body mass (Wu et al. 2012a). In the USA and EU,
the ingestion of melamine level above the safety limit is
2.5 ppm, and in China, it is 1 ppm. So, it is important to set
up a rapid, effective, and sensitive detection of melamine.
Nowadays, the main methods to detect melamine in milk
and dairy products are the following: high-performance liquid
chromatography (HPLC) (Peng et al. 2011), HPLC–mass
spectrometry, gas chromatography, ultra performance liquid
chromatography-mass spectrometry/mass spectrometry, and
capillary zone electrophoresis (Li et al. 2010). Additionally,
there are also some new methods to monitor melamine, such as
surface enhanced Raman spectroscopy, fluorescence, spectro-
photometric absorption, and chemiluminescence (Ding et al.
2010). Although these methods are highly sensitive, most of
1442
them are either time-consuming due to exhaustive pretreatment
or costly due to the expensive instrumentation. Therefore, the
simple, rapid, low-cost, easy operated methods are required to
develop for detecting melamine in milk and dairy products.
It is well known that gold nanoparticles (AuNPs) exhibit
visual sensing properties; the use of AuNPs as a colorimetric
signal depends on their colors of red or blue corresponding to
their dispersion or aggregation (Wu et al. 2011, 2012b).
According to this, several colorimetric systems have been set
up to detect proteins, ions, viruses, DNA, cancerous cells, β-
lactamase, and, of course, melamine. For detecting melamine,
the surface-functionalized AuNPs together with analyte can
transform from dispersive to aggregated; meanwhile, the color
of AuNPs changes (Guo et al. 2010). One research reports that
the color change induced by the triple hydrogen bonding recog-
nition between melamine and a CA derivative grafted on the
surface of gold nanoparticles can be used for reliable detection
of melamine (Han and Li 2010; Lee et al. 2010; Qi et al. 2010).
In some assays, electrostatic interactions between melamine
and citrate-stabilized AuNPs have been developed for the
detection. Actually, it is not only because of electrostatic
interaction that AuNPs can thus be cross-linked directly in
the presence of certain amounts of melamine (Araujo et al.
2012; Campbell et al. 2007; Roy et al. 2011). So, in this work,
the structure and reaction principle have been discussed be-
tween melamine and unmodified AuNPs. Moreover, based on
this, we optimized this rapid visual detection of melamine by
unmodified gold nanoparticles and hope to provide the basic
for modifying the AuNPs or linking with varied analytes in
order to improve the sensitivity for detection.
Experimental
Materials and Apparatus
Chloroauric acid, sodium citrate, and sodium chloride were
bought from Shanghai Chemical Reagent Company
(Shanghai, China). Melamine was obtained from Sigma-
Aldrich (Milwaukee, WI, USA). The raw milk was purchased
from a local cattle farm nearby. All reagents were of analytical
grade. Milli-Q water of 18 MΩcm was used in all experiment.
Absorption spectra were performed on a UV-2410 PC UV–
Vis Spectrophotometer (SHIMADZU) and a Bio-Tek Epoch
Microplate Reader (Bio-Tek Instrument, USA). The pH meas-
urements were recorded on DELTA 320 pH meter
(METTLER TOLEDO).
Preparation of AuNPs
Following usage, all glassware was cleaned in aqua regia-
prepared 3:1 HNO3/HCl, washed thoroughly in water, and
dried in the dry oven. AuNPs with a diameter of 18.0 nm were
Food Anal. Methods (2013) 6:1441–1447
prepared by the trisodium citrate reduction method (Li et al.
2010). One hundred milliliters of 0.01 % (w/w) HAuCl4 sol-
utions was heated until boiling. After 2 min, 3.5 mL of 1 %
sodium citrate solution was added quickly with vigorous stir-
ring. The color immediately changed from pale yellow to
purple, then to dark red, and to bright red in the end. After
about 15 min, the heating was stopped. The solutions were
stirred continuously about 15 min until it cooled to room
temperature. Finally, the cooled solution was diluted with
100 mL of water and stored in the refrigerator at 4 °C.
According to this procedure, we obtained nanogolds with
consistent particle size of 18 nm.
Principle of Detection of Melamine Using AuNPs
A series of analysis were performed to assess the interaction
between the AuNPs and melamine as follows: First, standard
solution of melamine was diluted into different concentration,
mixed with AuNPs as 1:1, and the final concentrations of
melamine were 0, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, and 10 μM
(1.0 μM = 0.126 ppm). The samples were sent to the
Instrumental Analysis Center of SJTU to find the melamine
concentration-dependent size increase in gold–melamine par-
ticle aggregation by dynamic light scattering (DLS) analysis.
Second, to understand the melamine aggregation mechanism,
we chose six different amino compounds (1.0 μM) mixed
with AuNPs (Chi et al. 2010). With pure water and melamine
as the negative control and positive control, we balanced
mixed sample and AuNPs for 5 and 300 min at room temper-
ature then quantified by the absorption ratio (A640/A520).
Third, AuNPs were premixed with NaCl of different concen-
trations (0, 10.0, and 20.0 mM) to detect melamine in 5 min.
Colorimetric Detection of Melamine
A typical colorimetric analysis was realized as follows: First, a
series concentration of melamine were mixed with AuNPs as
1:1, and the final concentrations of melamine were 0, 0.4, 0.6,
0.8, 1.0, 2.0, 4.0, and 10 μM. After reaction for 5 min, the color
change was observed by both the naked eyes and the spectro-
photometer scanned from 400 to 800 nm. Second, the mixture
mixed the same way as above was observed by both naked
eyes and the spectrophotometer scanned at 640 and 520 nm
after reaction for 0, 5, 10, 30, 60, 120, 300, and 600 min. Third,
the mixture of melamine (1.0 μM) and AuNPs was adjusted
from pH1 to pH14 and scanned by the spectrophotometer at
640 and 520 nm after reaction for 10 min.
Developing of Method
A series concentration of melamine were first added into raw
milk samples, and after pretreatment, they were mixed with
AuNPs as 1:1, and the final concentrations of melamine were
Food Anal. Methods (2013) 6:1441–1447
0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,
1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 μM. The samples were scanned
by the spectrophotometer at 640 and 520 nm after reaction for
10 min. After the standard curve was finished, the fortified
recovery test of melamine in raw milk was taken based on it.
Result and Discussion
Principle of Melamine Detection Using Unmodified AuNPs
as Colorimetric Probe
Figure 1 illustrated that melamine has a strong interaction
with AuNPs. When melamine exists, this interaction will
reduce the stability of melamine against aggregation and
cause AuNPs' visible color changes from red to blue in
few seconds. In addition, AuNP solution shows a particular
color because of the surface electron oscillations induced by
visible light of suitable wavelength, which is dependent on
interparticle spacing (Ai et al. 2009). According to the three
amine groups (–NH2) of melamine, it seems that AuNPs are
easily bind to amine functional groups.
The principle of DLS to measure the size of AuNPs was
based on the Brownian motion of particles that cause a
Doppler shift of incident laser light. In our research, this
method was performed to assess the interaction between the
melamine and AuNPs (Wei et al. 2010). The sizes followed
were based on the average of three repeated measurements.
Figure 2 showed a relationship between melamine concen-
tration and size increase in AuNPs–melamine particle ag-
gregation. The color changes from red to blue happened in
melamine concentration higher than 0.6 μM, as the average
hydrodynamic particle diameter of AuNPs–melamine was
larger than 60 nm. There were reports that aggregate size
and interparticle distances caused this surface plasmon
resonance-based colorimetric shift. So, with the melamine
Fig. 1 Schematic
representation of the
colorimetric detection for
melamine
1443
added more in AuNPs, the mixture color will change from
red to blue, and the average particles of melamine–AuNPs
will become greater, just as Fig. 2 reveals.
We compared the colorimetric difference of AuNPs to six
amino compounds, 1—ammonium hydroxide, 2—propyl-
amine, 3—1,2-cyclohexanediamine (Wei et al. 2010), 4—
poly-L-lysine (Liang et al. 2011), 5—adenine, 6—triazine,
and 7—melamine, to deeply find out the melamine-AuNPs
aggregation mechanism. For 5 min, there was no visible
color change of AuNPs with the addition of other six com-
pounds. Testing by the UV–Vis spectroscopic response of
AuNPs with the six compounds of 1 μM in solution (Fig. 3),
the A640/A520 ratios for 1 and 2 are the same as the blank.
Meanwhile, the A640/A520 ratios for 3, 4, 5, and 6 are a little
higher than the blank, which means that 3, 4, 5, and 6
change the interparticle distances or cause the aggregation
of melamine, but not obvious. For 300 min, there was still
no visible color change of AuNPs with 1 and 2, but the color
of AuNPs with amino compounds 3, 4, 5, and 6 changed
from red to purple when the blank was red and 7 was deep
blue. The A640/A520 ratios of 3, 4, 5, and 6 for 300 min
further prove that they can cause AuNP aggregation, but not
as much as melamine. There are no effects to AuNPs with 1
and 2. In conclusion, the exocyclic amino groups induce the
aggregation of AuNPs; the ring nitrogen atoms and the
symmetrical structure of compounds will make this aggre-
gation obvious.
As we mentioned above, the interaction between mela-
mine and AuNPs led to the aggregation of AuNPs with the
colorimetric signal change. Wei et al. (2010) pointed out
earlier that the sensitivity and dynamic range of the colori-
metric signal are dependent on the resistance of AuNPs to
aggregation and could be adjusted by changing the buffer
composition of the AuNPs. According to this, we chose
sodium chloride to investigate this inference because sodium
chloride is famous to destabilize the AuNPs. AuNPs were
1444
Fig. 2 DLS demonstration of melamine concentration-dependent
increases in particle size
premixed with NaCl of 0, 10.0, and 20.0 mM to detect mela-
mine (Fig. 4). As Fig. 4 illustrated, the presence of NaCl in this
system of melamine–AuNPs obviously amplified the signals
of sensitivity and dynamic range. If we can figure out the
correct relationship between the addition of NaCl and the
change of melamine–AuNP signals, we can pretreat the
AuNPs so that a significant color change can happen around
the safety level when we want to determine whether there is
melamine above the safety level in the samples.
Colorimetric Detection of Melamine
UV–Vis Absolution Spectra of AuNPs As reported previously,
melamine has a strong electrostatic interaction with citrate-
stabilized AuNPs, which decrease the stability of AuNPs
and cause the visible color changes (Chi et al. 2010). There
are still problems using this character as a probe to indicate the
Fig. 3 The A640/A520 ratios of AuNPs with the addition of seven amino
compounds with different concentration
Food Anal. Methods (2013) 6:1441–1447
Fig. 4 The absorbance ratio of AuNPs–melamine in the presence of
NaCl
existence of melamine. Figure 5 shows the color changes and
UV spectra of melamine–AuNPs after reaction for 5 min. The
blank group shows that citrate-stabilized AuNPs are red be-
cause of their surface plasma resonance at 520 nm. Along
with the increase of melamine added in the mixture of
AuNPs–melamine, the solution color changes from wine
red, purple to blue progressively, and the surface plasma
resonance at 640 nm increases; on the contrary, the
surface plasma resonance at 520 nm decreases. For
5 min, clear color changes were observed to purple, blue at a
melamine final concentration 2.0 μM. Meanwhile, the aggre-
gation of AuNPs can be monitored by UV–Vis spectroscopy
at a melamine final concentration of 0.8 μM. From Fig. 5, we
can see that more melamine will cause more aggregation of
AuNPs and the significant aggregation of nano particles in the
presence of more than 2.0 μM (252 ppb) melamine for reac-
tion 5 min.
Fig. 5 Visual color change and the spectrophotometric demonstration
Food Anal. Methods (2013) 6:1441–1447 1445

Fig. 6 The absorption ratio for different concentration of melamine in
different time
Time Specificity About Aggregation of AuNPs We also chose
eight groups to examine the aggregation of AuNPs in the
presence of melamine for 0∼600 min in order to evaluate the
aggregation kinetics of AuNPs. As shown in Fig. 6, the
higher the concentration of melamine is, the faster the
AuNPs aggregate. When there is a high melamine concen-
tration (>1.0 μM), the extinct ratio rises fast in the initial
stage. At the concentration of 1.0 μM, the absorption ratio
(A640/A520) begins to increase from original 0.108 to 0.118
obviously during the first 10 min with the mixture color
changed from wine red to light purple. At the high concen-
tration (>1.0 μM), the ratio increases more obviously during
the first 10 min, with the color changed from wine red to
dark blue. At the concentration of 0.6 and 0.8 μM, the ratio
begins to increase during 10 to 30 min, and the mixture
color will become light blue, not dark blue at last. It means
not all the particles of AuNPs get aggregated at this concen-
tration of melamine. At the concentrations of 4.0 and
Fig. 8 The calibration curves of melamine in raw milk
10.0 μM, the ratio decreases after reaction for 120 min. It is
because all the particles of AuNPs get aggregated and depos-
ited, and the solution color becomes clear from dark blue. In
conclusion, it is enough for us to detect the low concentration
of melamine (<1.0 μM) by absorption ratio after reaction for
10 min. We do not need more obvious result (after more than
30 min) because rapidity is also important.
The Optimization of pH The effects of pH can be serious to
the interaction of AuNPs with small molecule. As we know,
melamine is alkalescence with the pKa of 5.05 (Liang et al.
2011); the pH of solution also affects the solubility of
melamine. We fix the melamine concentration at 1.0 μM
and adjust the acetic acid buffer solution with hydrochloric
acid and sodium hydroxide. Figure 7 shows that the pH of
solution hardly affects the aggregation of AuNPs, and the
absorption ratio (A640/A520) is very low in strong base media
and strong acidic media (pH>12.0, pH<2.0). It is because
melamine can be transformed into cyanuric acid losing three
amino groups in strong basic media and strong acidic media

Table 1 Application of this method and HPLC to detect the melamine

in raw milk samples spiked with different amounts of melamine

Sample Concentration of melamine (ppm) Recovery (100 %)

Added Found (AuNPs)
1 0.200 0.24 (±0.01) 120
2 0.500 0.46 (±0.02) 92
3 1.000 1.05 (±0.05) 105
4 2.000 1.98 (±0.10) 99
Sample Concentration of melamine (ppm) Recovery (100 %)
Added Found (HPLC)
1 0.200 0.21 105
2 0.500 0.51 102
3 1.000 1.00 100
4 2.000 1.99 99.5
Fig. 7 Effect of pH on the absorption ratio (A640/A520)
1446
(Chi et al. 2010), and cyanuric acid cannot lead to the
aggregation of AuNPs. At pH8.0, the absorption ratio is
the highest during the pH1.0 to 14.0, thus we chose 7.0 as
the best pH for the detection media.
The Calibration Curves of Melamine in Raw Milk To dem-
onstrate whether the citrate-stabilized AuNPs can be used
for the detection of melamine in milk powder, we added
different amounts of melamine to the raw milk before sam-
ple pretreatment. Milk is a complex system and its various
components serve as potentially interactive species with
melamine regardless of detection methodology. For the
casein-based milk, the casein exists in milk with the form
of water-soluble calcium salt of a phosphoprotein; acid
treatment removes the calcium cation, leaving a water-
insoluble phosphoprotein (Li et al. 2010). So, we add acetic
acid into the milk and centrifugate the samples to separate
the liquid component from the white, opaque precipitation.
Then, we adjust pH to 8.0 and supply the solution volume
with pure water.
When the concentration of melamine is high (>2.0 μM),
the absorption ratio increases too fast for us to control and
the process of color shift is hard to observe clearly in the
first 10 min. So, we chose the concentration range of
0∼2.0 μM to evaluate the melamine concentration by com-
paring the A640/A520 value. Beyond the concentration range,
the samples can be diluted into this range probably and
detected. As shown in Fig. 8, melamine in the raw milk
samples could be quantified within the range of 0.6 to
2.0 μM, and the detection limit is less than 0.6 μM (the
final concentration). Since ingestion of melamine at levels
above the safety limits are 2.5 ppm (20 μM) in USA and EU
and 1 ppm (8 μM) for infant formula in China, these
calibration curves of melamine can be used to detect the
melamine in raw milk samples.
Detection of Melamine in the Raw Milk Samples Before sam-
ple pretreatment, we added different amounts (0.2, 0.5, 1.0,
and 2.0 ppm) of melamine to the raw milk. This proposed
method and HPLC were applied to analyze melamine in the
raw milk samples. As it shown in Table 1, the recoveries are
between 90 and 120 % by AuNPs, while the recoveries are
between 99 to 105 % by HPLC. It indicates that this method
using AuNPs as a probe for rapid detection of melamine in
raw milk is feasible.
Conclusions
In summary, a simple and rapid colorimetric assay for mela-
mine has been demonstrated. Due to the interaction between
AuNPs and melamine, the AuNPs can be induced from
Food Anal. Methods (2013) 6:1441–1447
dispersion to aggregation, meanwhile the color changes from
red to blue. This proposed detection of melamine can be taken
after simple pretreatment and the detection limit is far less than
the safety limits in China. The whole analysis only needs about
20 min including pretreatment, can be observed by the naked
eye, and simple to operate without sophisticated instruments.
So, this cheap method will probably be used to detect mela-
mine in other foods. What is more, in this work, we explore the
interaction principle between melamine and citrate-stabilized
AuNPs further and establish the foundation for modifying the
AuNPs and optimizing the system of AuNPs–melamine in the
future.
Acknowledgments This work was supported by the National High-
Tech Research and Development Plan (2012AA101405), the Special
Fund for Agro-Scientific Research in the Public Interest of China
(200903056), the National Natural Science Foundation of China
(31071860), and the National key Technology R&D Program
(2010BAK69B18).
Conflict of Interest There are no conflicts of interest.
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