Article

pubs.acs.org/ac

Melamine Sensing in Milk Products by Using Surface Enhanced

Raman Scattering
Ansoon Kim, Steven J. Barcelo, R. Stanley Williams, and Zhiyong Li*
Cognitive Systems Lab, Hewlett-Packard Laboratories, 1501 Page Mill Road, Palo Alto, California 94304, United States
S Supporting Information
*

ABSTRACT: Simple and rapid detection of trace amounts of melamine in milk

products has been achieved with a portable sensor system based on surface-enhanced
Raman scattering (SERS). The sensor system comprised high-performance gold
nanonger SERS sensor chips and a custom-built prototype portable Raman
spectrometer. Compared to the FDA procedure and previously reported studies that
were limited to laboratory settings, our sampling and analytical methods are simple
(with one sampling step), less time-consuming, and cost-eective. We found the limit
of detection (LOD) of the melamine is 120 parts per trillion (ppt) in water and 100
parts per billion (ppb) in infant formula, which are well below the FDAs tolerance
level of 1 ppm in infant formula.

technique extremely appealing as real-time sensors for eld

application. However, the lack of reliable, uniform, low cost,
and ease-of-use SERS enhancement structures has prevented
the wide adoption of this technique for general applications.
While there are a few recent reports on detecting melamine in
milk or formula using SERS, either sample pretreatment by
centrifugation6,22,23 or an extended sampling time of 24 h were
needed in order to obtain sucient sensitivity.24 One of the
main performance limitations in those reports was the
insucient enhancement of the SERS structures available in
the studies. Here we develop a novel gold nanonger SERS
structure and demonstrate melamine sensing for which only a
simple ltration step is needed to remove large molecules such
as proteins in milk or infant formula samples before sensing for
melamine. The ltered sample solution is exposed directly onto
a SERS chip with periodic nanonger structures, followed by
rinsing with ethanol and drying in air. Finally, as shown in
Figure 1b, Raman spectra can be measured by inserting the
SERS chip into the sample slit in a custom-built portable
spectrometer. The entire process can be completed within less
than 20 min including sample preparation, which makes it
highly practical for eld deployable sensing applications.
In this paper, we describe the development of the sampling
procedure for rapid melamine sensing in milk and infant
formula. We also present here the characterization of melamine
bonding on our gold nanonger surfaces and the eect of
nanonger closing toward SERS performance. Melamine
sensing in infant formula using laboratory and portable
Raman spectrometers is compared.

elamine is a nitrogen-rich compound mainly used to

produce kitchenware, commercial lters, ame retardants, and other products, and its wide use may result in the
presence of trace amounts of melamine in the food supply.1
Melamine was implicated in pet and human food recalls in
20071,2 and in the global food safety alarm in 2008 involving
milk and infant formula.3,4 Since milk products contaminated
with melamine can lead to kidney disease and even mortality in
infants, both the United States and China have set a threshold
of 1 part per million (ppm) of melamine in infant formula. For
all other foods, less than 2.5 ppm is considered low risk.1,5,6
Liquid chromatography/tandem mass spectrometry,7 highperformance liquid chromatography/mass spectrometry
(HPLC/MS),8,9 gas chromatography/mass spectrometry
(GC/MS),10,11 and enzyme-linked immunosorbent assay
(ELISA)12 are typically used to detect melamine in food
products in laboratories. These methods require timeconsuming sample preparation steps after samples are transported to a laboratory and can only be done by highly trained
operators on expensive instruments, which makes the method
impossible for eld test applications. A standard FDA
procedure13,14 for melamine testing in milk or infant formula
is illustrated in Figure 1a. Isotope-labeled melamine is rst
added to a test sample, followed by extracting the sample with a
5050 mixture of acetonitrile and water. The extracts are
centrifuged and then separated using solid phase extraction.
Finally, the concentration of melamine in the sample is
analyzed using a high-performance liquid chromatography/
mass spectrometer (HPLC/MS).
Surface-enhanced Raman scattering (SERS), on the other
hand, has long been projected as a powerful analytical
technique for chemical and biological sensing applications.1517
Pairing with portable Raman spectrometers1821 makes the
2012 American Chemical Society

Received: July 23, 2012

Accepted: October 8, 2012
Published: October 8, 2012
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Figure 1. Comparison of melamine sensing in milk using (a) a standard FDA procedure versus (b) our nanonger-based sensing solution. The
representative SEM images of open (left) and closed (right) pentamer gold nanongers before and after treatment with the ltered milk,
respectively, are shown along with the illustration of how the gold nanonger chips were used for molecular sensing. The photographs show the
components of the prototype portable sensor system, which include the custom-built portable reader and the pentamer nanongers chips, shown
both unmounted and mounted on an aluminum strip.

EXPERIMENTAL DETAILS
Preparation of Gold Nanonger Chips. As described in a
previous report,25 the polymer nanonger structures were
fabricated on Si wafers using nanoimprint lithography (NIL).
The typical diameter of each nanonger was 140 nm and the
height was 530 nm. Au with nominal thickness of 70 nm was
deposited over the polymer nanongers by e-beam evaporation
to produce the gold nanonger substrates. The substrates were
then diced into chips of 5 mm 5 mm size (Figure 1b) and
used for sensing either directly or mounted on strips.
Sampling Procedures. Melamine was dissolved in
deionized (DI) water to prepare a 1000 ppm stock solution.
The melamine solution was spiked into whole milk and infant
formula purchased from a local grocery store. The formula
solution was prepared following the instructions on the label.
For sample ltration using dialysis, 100 L of either milk or
formula solution spiked with melamine was introduced into a

Slide-A-Lyzer MINI Dialysis Device (2K MWCO, Thermo

Scientic), followed by dipping the device into 1.2 mL of DI
water for 10 min. The water solution after the dialysis was
exposed to the goldnanonger chips for 10 min. For the gel
ltration method, 400 L of formula solution with various
levels of spiked melamine was loaded into a gel ltration
column (NAP-25, GE Healthcare) that was packed in a 10 mL
syringe. The equilibration step before introducing the formula
solution into the column was skipped to simplify the process.
Puried formula sample was eluted with DI water and collected
in 500 L portions. Each portion was exposed to a gold
nanonger chip for 10 min. The gold nanonger chips were
then air-dried, followed by rinsing with pure ethanol to ensure
gold nanonger closing on the chips for Raman measurement.
No Raman peaks from the storage buer solution in the
column were observed. The intensity of the melamine Raman
signal peaked at the 28th and 29th portions of eluted solutions.
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Raman Measurements. Raman measurements were

performed either using an upright confocal Raman microscope
(Horiba Jobin Yvon T64000) equipped with a nitrogen-cooled
multichannel CCD detector or a custom-built portable Raman
spectrometer. For the laboratory Raman spectrometer, a 784.6
nm solid state laser was used as the excitation source with a
measured power of 300 W at sample surfaces with a focal spot
of about 2 m in diameter. All spectra obtained from this
spectrometer were collected with the same setup through a
100 objective lens and recorded with a 5 s accumulation time.
The custom-built portable Raman spectrometer, shown in
Figure 1b, is equipped with a 785-nm diode laser for
illumination over an area about 100 m in diameter on the
sample surface. Melamine sensing was achieved using a laser
power of 10 mW with 1 s integration time. The instrument
provides Raman spectra over the range of 4501780 cm1. The
spectral resolution of this instrument is 1012 cm1.

pentamer nanonger chips even for high melamine concentration solution (1 mM) in water was not expected initially.
However, with scanning electron microscopy (SEM), we
noticed that the pentamer nanongers were not fully closed
on the chips (as shown in the blue square inset in Figure 2)
after the simple air drying following the soaking process. In our
previous study,27 we found that ethanol-soaked pentamer
nanongers were fully closed after drying. Therefore, we then
rinsed the melamine-exposed chips with ethanol and
subsequently dried in air. By doing the additional ethanol
rinsing, we found the nanongers are fully closed afterward as
shown in the SEM image in the red square inset in Figure 2. As
a consequence, the Raman spectrum obtained from these fully
closed nanonger chips shows greatly improved melamine
signals, with more than a 20-fold increase of the major peak at
710 cm1. This further supports that nanonger-closing and
molecule trapping25,26 between the nanonger tips are critical
to the SERS sensing performance. The clustering of high aspect
ratio pillar structures is generally caused by the capillary force
between neighboring pillars, when liquid wets and forms a
meniscus between them.30,31 However, since the polymer
pillars in our nanonger structures are hydrophobic, ethanol is
likely able to wet the surface of the nanongers better than
water. Therefore, the lateral capillary force between the
nanongers will be stronger for the sample rinsed with ethanol,
causing more consistent nanonger closing. Hence, we have
subjected the nanonger chips with the additional ethanol
rinsing for improved and reliable sensing performance for the
rest of the study.
By comparing the Raman spectra measured from melamine
powder6,23,28,29,32,33 and melamine-trapped nanongers, we can
understand the interaction of melamine molecules with gold
nanongers. The Raman spectrum of the melamine powder
(Figure S1) and the peak assignments of the spectrum (Table
S1) are supplied in the Supporting Information. The most
intense peak in the powder spectrum was observed at 675 cm1
and assigned to the in-plane ring breathing II mode, which
involves an in-plane deformation of the triazine ring with the
vibration of the amino nitrogen atoms. The other strong
Raman band at 984 cm1 is the in-plane ring breathing mode I
of the triazine ring related to the ring nitrogen atoms. The
Raman spectrum of the nanonger trapped melamine showed
two specic melamine peaks at 710 cm1 and 985 cm1. The
peak at 710 cm1 had red-shift about 35 cm1 from that of
melamine powder. On the other hand, the ring-breathing mode
I at 985 cm1 had almost no shift. This observation suggests
that the melamine interacts with the gold nanonger surfaces
through the amino groups rather than the trazine ring.
In order to characterize the nanonger chips for quantitative
melamine sensing, we prepared dierent melamine water
solutions with concentrations from 1 nM to 1 mM and
introduced them to the periodic pentamer nanonger chips for
SERS measurement. Figure 3a shows the SERS spectra of
dierent concentrations of melamine, measured from the
periodic pentamer nanongers. The spectra indicate that the
characteristic melamine peaks are clearly observed at a
concentration of 1 nM and saturate at around 10 M. The
intensity of the characteristic peak of melamine at 710 cm1 as a
function of the melamine concentration was plotted in Figure
3b for melamine sensing on periodic pentamer nanongers.
The error bars were obtained from at least two measurements
on three dierent locations on the same chip as well as on three
dierent chips.

RESULTS AND DISCUSSION

Previously, we reported that the exibility and high aspect ratio
of the gold nanongers allowed them to self-close during
evaporation of solutions.2527 Free standing pentamer nanongers arranged in 5-fold symmetry identied previously as
providing the highest signal enhancement for trans-1,2-bis(4pyridyl)-ethylene (BPE)27 are shown in Figure 1b. After being
exposed to the ltered milk samples, the gold nanongers
collapse into well-dened groups and form self-limiting sub nm
gaps between the gold nanonger tips.26 The molecules thus
being trapped among the touching gold nanonger tips will
experience greatly amplied electromagnetic elds under
incident laser illumination and hence generate uniform, strong
Raman signals for molecule identication and quantication.26
In order to understand the interaction of melamine with the
gold nanonger surfaces and optimize the method of applying
the gold nanonger chips for melamine sensing, we studied the
melamine in water rst. We prepared a 1 mM melamine
solution in DI water and dipped the pentamer nanonger chips
in the solution for 10 min. The rst drying method we used
following the dipping process was simply air-dry. A
representative Raman spectrum collected from the chips with
this air-dry method was shown in the blue graph in Figure 2,
where very weak melamine signals6,23,28,29 were observed at 710
and 985 cm1. The weak melamine signal acquired on the

Figure 2. Eect of the nanonger closing on the sensing performance.

The SERS spectra and SEM images (scale bar, 500 nm) were
measured from the melamine solution exposed pentamer nanongers
before (blue) and after (red) rinsing the chip in pure ethanol and
drying to fully close the nanongers.
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the surface (negatively cooperative binding) and k = 4.6 107.

At concentrations above 10 M, the SERS intensity reached a
plateau indicating the adsorption of the molecules saturated the
surfaces. At low concentrations, where c k, a loglog plot of
ISERS vs c revealed a linear relationship (log ISERS n log c +
const), as shown in the inset of Figure 3b for the experimental
data at concentrations below 100 nM. This suggests that
pentamer nanonger chips can be used as reliable sensors for
quantitative analysis of melamine at low concentrations in water
solution. As a comparison, we also studied a dense nanonger
chip with random nanonger assemblies for quantitative
melamine analysis, which was described in the Supporting
Information. The limit of detection measured from the
pentamer nanonger chips was about 2 orders of magnitude
lower than that from dense nanonger chips. This is mainly due
to the fact that the pentamer nanongers can produce uniform
hot spots with greater eld enhancement than the random hot
spots formed on the dense nanonger arrays.
After calibrating melamine sensing in DI water samples, we
analyzed melamine in real milk samples. We spiked a whole
milk sample purchased from a local grocery store with 100 ppm
of melamine and tested the sample with pentamer nanonger
chips initially with the same method as used for the water
solutions. However, no melamine peaks were observed, mostly
due to the preferential interaction of milk components (e.g.,
proteins, fats, or lactose) with the nanonger surfaces. In
particular, proteins usually have chemically reactive functional
groups such as primary amine and sulfur linkages, which prefer
to bond to the gold nanonger surfaces and therefore block
melamine molecules from binding to the gold. In order to
detect melamine in milk, sample pretreatment is necessary to
lter out those large molecules, such as casein, other milk
proteins, or fat. A common technique in a laboratory
environment to pretreat milk is to use centrifugation to
separate the large molecules from the solution. However,
because a centrifuge is neither portable nor low cost, we
excluded this method from our sensor system. Instead, we
developed a dialysis method using a commercially available
mini dialysis kit from Thermo Fisher. Milk samples (0.1 mL)
spiked with melamine were pipetted into the dialysis well
containing a membrane with a pore size of 2 kDa molecular
weight cuto (MWCO). Then, the well was soaked in 1.2 mL
of DI water for 10 min to dialyze the sample. After the dialysis,
the pentamer nanonger chips were dipped into the clear water
solutions outside the well for 10 min.
Figure 4 shows the SERS spectrum of 1 ppm melamine in
water and the concentration-dependent SERS spectra of milk
samples spiked with melamine. The Raman intensity measured
from the water sample was reduced by half for the comparison
purpose. The characteristic melamine peaks were observed
from the dialysis ltered solutions. The most intense melamine
peak at 710 cm1 was monitored to determine the LOD of
melamine in milk. As shown in the spectra, the Raman intensity
of the melamine peak increased with melamine concentration
between 0 and 100 ppm. We found that the dialysis ltration
enabled us to detect the melamine concentration in milk as low
as 1 ppm. The dialysis procedure diluted the melamine
concentrations from the original milk samples (100 L) to
water (1.2 mL) by a factor of at least 13. The fact that our
SERS procedure still successfully detected the presence of
melamine in the dialyzed and diluted solution from milk
samples of 1 ppm melamine shows that our sensor system was
able to detect less than 80 ppb melamine in the dialyzed

Figure 3. Quantitative data of melamine sensing on pentamer

nanonger chips. (a) SERS spectra of melamine in water at dierent
concentrations acquired from pentamer nanonger chips. (b)
Quantitative curves of melamine sensing on pentamer nanonger
surfaces. The Raman intensity measured at 710 cm1 was plotted as a
function of melamine concentration. The error bars were obtained
from multiple measurements on dierent surface areas and samples.
The red curves are a t to the experimental data using Hills equation.
Loglog plot of the Raman intensity versus the concentration of
melamine is shown in the inset. The red line is a linear t to the
experimental data at low concentrations below 100 nM.

The interaction of melamine with pentamer nanonger

surfaces was further analyzed by tting the curve with the Hill
equation.34 Generally, the SERS intensity (ISERS) is proportional to the surface coverage of adsorbed molecules on hot
spots (), assuming all hot spots are uniform over the surface,
and the surface coverage follows the Hill equation, eq 1, as
shown below:
ISERS = V = V

cn
k + cn
n

(1)

where V is a constant, c is the concentration of the molecular

solution, k is the equilibrium constant for dissociation, and n is
a cooperative constant. When n > 1 or n < 1, surface-bound
molecule increases or decreases, respectively, the anity of the
incoming molecules to the surface. Note when n = 1, the Hill
equation is equivalent to the Langmuir isotherm, where the
binding anity of the molecule to the surface is not dependent
on the binding occupancy on the surface. We tted the
quantitative curve measured from the pentamer nanonger
chips and obtained n = 0.55, which indicates that surface-bound
melamine decreases the anity of the incoming molecules to
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to the nutrient ingredients added to infant formula, which

interfered with melamine sensing. Therefore, the dialysis
method was not sucient to enable the detection of the
melamine in the infant formula. A dierent method based on
gel ltration chromatography was developed to detect
melamine in the formula. Inexpensive and portable gel ltration
columns were selected to separate the components in the
formula based on their size. Small molecules such as melamine
enter many pores in the gel, and so travel slowly, and are eluted
later. On the other hand, large molecules like proteins, fats, or
sugars enter few pores and so travel rapidly. After the formula
solution was introduced to the gel column, we used DI water to
elute the column and collected the eluted solution into portions
of 500 L each, which were then analyzed as described in the
previous section. We obtained the maximum melamine peaks in
the 28th and 29th portions. All the results shown here were
measured from the 28th portion. Figure 6 shows the SERS

Figure 4. SERS spectra at dierent concentrations of melamine in milk

after being treated with the dialysis ltration method. All the data were
acquired by using pentamer nanonger chips.

solutions. However, there was still a signicant discrepancy

between the detection limit of melamine in DI water described
in the previous sections and in the dialyzed milk samples
presented here. This may be due to a dialysis time shorter than
the 24 h suggested in the instructions of the dialysis kit. In
addition, even though the dialysis method eectively removed
all the large molecular weight species from the milk samples,
there were still many low molecular weight species below the
MWCO of the membrane that passed through and competed
with the melamine molecules for adsorption in hot spots on the
nanonger surfaces. Furthermore, melamine may interact with
proteins in milk to form complexes through hydrogen bonds
between the amino groups on melamine with proteins and
subsequently removed from the milk during dialysis to reduce
the actual melamine concentration in the dialyzed solution. We
believe there is still potential to further improve the detection
limit of melamine in milk solutions by optimizing the dialysis
procedure, such as the choice of membrane material and the
dialysis conditions.
In addition to the raw milk sample, we have also studied
melamine sensing in infant formula using the same dialysis
method described above. The result shown as the red graph in
Figure 5 is a SERS spectrum measured from 1 ppm of
melamine in formula after dialysis ltering. Compared to the
milk sample (black graph in Figure 5), we observed more peaks
and a large background in the formula sample. This is likely due

Figure 6. SERS spectra of melamine in infant formula separated by gel

ltration. The inset shows the plot of Raman intensity measured at 710
cm1 as a function of melamine concentration. The error bars were
obtained from multiple measurements on dierent surface areas.

spectra of melamine spiked in formula at dierent concentrations. Considering the high signal-to-noise ratios in the
spectra, the results depict clear dierences between the spectra
of the control (0 ppb) and the formula samples spiked with low
concentrations of melamine (from 100 ppb to 1 ppm). The
characteristic melamine peaks around 710 and 1240 cm1
appear in the formula samples spiked with melamine, but no
melamine peaks appear in the pure formula sample. The inset
(Figure 6) shows the plot of Raman intensity measured at 710
cm1 versus melamine concentration. The error bars were
obtained from multiple measurements at dierent surface areas.
The limit of detection (LOD) for melamine sensing in the
infant formula using the gel ltration method is 100 ppb, which
is 1 order of magnitude lower than the FDA regulated level in
infant formulas and is also better than the LOD (250 ppb) of
the FDA standard procedure.14 Using the same gel ltration
method, we also found the limit of detection for melamine in
whole milk is 100 ppb (supplied in the Supporting
Information). In a previous report for melamine sensing in
infant formulas,24 Lee et al. observed a strong peak at 730
cm1 associated with one of the formula ingredients. We
observed no peaks around 730 cm1 from the control sample,
possibly because of the complete exclusion of this particular
species by the ltration process. Lee et al. reported a sensitivity

Figure 5. SERS spectra of 1 ppm melamine in milk (black) and infant

formula (red) ltered by using the dialysis method.
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of 200 ppb on roughened gold substrates for milk samples

without any pretreatment, but an extended sampling time of 24
h was required to achieve the reported sensitivity. Our method
with the LOD of 100 ppb achieved within 20 min of sample-todata response time holds great potential for simple and rapid
detection of melamine in milk products.
Finally, the quality of the Raman spectra acquired on our
portable reader was checked against a laboratory Raman
spectrometer. Figure 7 shows the Raman spectra of 1 ppm

on either dialysis or gel-ltration was easy to use and fully

compatible for eld applications in a limited-resource environment, such as for consumer applications. Using the gel ltration
method, we found the limit of detection for melamine in infant
formula and whole milk is 100 ppb, which is well below the
FDA regulated level of 1 ppm in infant formulas. The
demonstration of the high performance of our portable sensor
system for melamine sensing opens new opportunities for
developing other applications that can provide simple, rapid,
and inexpensive chemical and biological sensing.

ASSOCIATED CONTENT

S Supporting Information
*

Additional materials about the Raman peak assignment, the

dense nanonger chip study, and melamine sensing in whole
milk using the gel ltration method. This material is available
free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Phone: +1-650-236-4393. E-mail: zhiyong.li@hp.com.

Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
This work was partly supported by DARPA.

Figure 7. SERS spectra of 1 ppm melamine in formula using the

portable SERS (red) and laboratory Raman spectrometer (black). The
Raman intensities measured at 710 cm1 using both spectrometers
were normalized .

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melamine in infant formula obtained from our custom-built

portable reader (red) and a JY research grade laboratory Raman
spectrometers (black) by using the gel ltration method
described above. SERS strips mounted with gold nanonger
chips after being exposed to the sample solutions and
subsequently dried were simply inserted into the sample slit
in the portable reader (shown in Figure 1b) and scanned with 1
s integration time. For the JY spectrometer, we positioned the
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obtained using both spectrometers. However, the portable
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laboratory spectrometer. Considering that only 1 s integration
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was considered to be sucient for melamine sensing
application. This indicates that a simple-to-use, eld-portable,
and cost-eective sensor system was successfully demonstrated
for trace level melamine sensing in milk products.

CONCLUSIONS
We developed a portable sensor system that can detect a trace
amount of melamine based on surface enhanced Raman
scattering with gold nanonger structures. Using the high
performance and reliable gold nanonger SERS chips, we have
achieved the limit of detection (LOD) of 120 parts per trillion
for melamine in DI water without any sample pretreatment. For
melamine sensing in commercial milk products, we have
successfully developed a simple and rapid protocol to detect
trace level melamine below the FDA regulated level using our
portable sensor system. The one-step sampling process based
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