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Direct SERS detection of contaminants in a complex mixture: rapid, singlestep screening for melamine in liquid infant formula†

Jordan F. Betz,ab Yi Chengb and Gary W. Rubloff*bc

Received 12th September 2011, Accepted 13th December 2011

DOI: 10.1039/c2an15846a

Melamine can be detected in infant formula without the need for

additional sample preparation or purification using a simple galvanic

displacement reaction to fabricate portable silver SERS substrates.

The reaction is rapid, inexpensive, and robust enough to perform

well on highly heterogeneous commonmetal objects such as tape and

coins.

The ability to detect contaminants in complex mixtures is of great

importance to many fields. Blood, urine, saliva, food, and environ-

mental samples are examples of important complex mixtures that

contain a variety of compounds of interest to the analytical field.

Traditional analytical approaches involve separation and purification

of the compound of interest from the other analytes in the matrix

prior to detection or quantification. A simple method to detect

analytes in complex mixtures without the need for separation and

purification steps would reduce the time, complexity, and cost of

analysing complex samples.

Melamine, a toxic chemical that is 66% nitrogen bymass, has been

added to pet food, milk, infant formula, and other foodstuffs to

increase the apparent protein content of the food since the most

common test for the protein content of a food uses the amount of

nitrogen as a proxy for the amount of protein in a sample of food.

Melamine contaminated foods (most notably infant formula) have

cause severe health problems in China, including renal failure and

even death,1,2 and thus melamine levels in food are regulated by

several governing bodies worldwide.

Surface enhanced Raman spectroscopy (SERS), a non-destructive

spectroscopic technique, has been applied to the detection of low

concentration analytes in many fields. Here we demonstrate the

SERS detection of a toxic food contaminant (melamine) in a complex

mixture (commercially available infant formula). A handful of

groups have previously reported the SERS detection of melamine

aFischell Department of Bioengineering, University of Maryland, CollegePark, MD, USAbInstitute for Systems Research, University of Maryland, College Park,MD, USA. E-mail: rubloff@umd.edu; Fax: +01 301-314-9920; Tel: +01301-405-3011cDepartment of Materials Science and Engineering, University ofMaryland, College Park, MD, USA

† Electronic supplementary information (ESI) available: Additionalscanning electron micrographs of the Cu tape substrate. See DOI:10.1039/c2an15846a

826 | Analyst, 2012, 137, 826–828

purified from food samples,3–6 but these typically require time-

consuming preparation methods such as solvent extraction and

expensive commercial SERS substrates. To our knowledge, only one

other group has reported the SERS detection of melamine contam-

ination in food samples without using separation methods,7 and this

required soaking the substrate in the melamine solution for 24 h to

detect melamine contamination below 100 parts per million (ppm).

This report is the first to combine rapid SERS detection of melamine

contamination in food without the need for purification or additional

equipment while also making use of a simple, inexpensive, and highly

portable SERS substrate.

In recent years, galvanic displacement has received increasing

attention as a simple method to form highly effective SERS

substrates.8–12 This spontaneous electrochemical reaction forms

fractal micro- and nanoscale structures on a surface that yield

excellent SERS enhancement. The galvanic displacement of Cu by

Agwas used to createAgmicro- and nanostructures on two common

low-cost and highly portable surfaces- Cu tape and a Cu coin

(U.S. penny). In brief, a solution of 5 mMAgNO3 was placed on the

surface of the tape or coin and allowed to react for 5min before being

dried using nitrogen gas. A 2 mL volume of commercially available

infant formula adulterated with different amounts of melamine was

placed on the surface and a spectrumwas acquired immediately using

a 785 nm diode laser, which was used to evaluate the possibility of

using these substrates for portable, point-of-sampling analysis. Ten

points on the substrate were scanned with a total signal acquisition

time of 150 s (3 s acquisitions averaged 5 times per spot over 10 spots)

for the 1000 ppm, 100 ppm, and 10 ppm levels and 500 s (10 s

acquisitions averaged 5 times per spot over 10 spots) for the 5 ppm,

1 ppm, and 0 ppm (normal infant formula) levels.

Fig. 1 shows the spectra acquired from the Cu tape substrate

(Fig. 1a) and the Cu coin substrate (Fig. 1b). Melamine has a strong

characteristic peak corresponding to an in-plane deformation of the

triazine ring4,5,13 peak reported at 676–690 cm�1, depending on

reaction conditions. This peak was used to identify melamine

contamination of the formula, given the lack of strong peaks from the

infant formula in this spectral region as can be seen in the 0 ppm cases

in Fig. 1. Both the Cu tape and coin substrates enable the detection of

melamine contamination down to the 5 ppm level, which can clearly

be distinguished from normal, uncontaminated infant formula

(0 ppm melamine) based on the presence of the peak at approxi-

mately 685 cm�1 for the Cu tape and 678 cm�1 for the coin substrates,

henceforth referred to as the 680 cm�1 peak for simplicity. At 1 ppm,

This journal is ª The Royal Society of Chemistry 2012

Fig. 1 Average SERS spectra of melamine adulterated commercially

available infant formula on Ag substrates formed on (a) Cu tape and (b)

a Cu coin. The peak centred around 680 cm�1 is due to an in-plane

deformation of the triazine ring of melamine, which can clearly be

distinguished down to the 5 ppm levels in both cases. Spectra are offset to

better show the characteristic melamine peak.

Table 1 Coefficients of variation for the substratesa

Substrate Type Sample CVSubstrateCV Point CV

Cu Tape 0.506 0.702 N/ACu Coin 0.534 0.998 0.385

a Sample CV is the coefficient of variation for 10 different spectraacquired within the same sample droplet. Substrate CV is thecoefficient of variation between five different substrates each with tenspectra. Point CV is the coefficient of variation for five differentsamples tested on the same coin substrate. The CV was calculated byintegrating the area of the characteristic peak for melamine in the 1000ppm case and dividing the standard deviation of these areas by themean of the areas.

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the samples were indistinguishable fromnormal formula solely on the

basis of the average spectra and the average integrated peak area.

Comparing these results with the only other published instance of

direct SERS detection of melamine contamination in milk without

any pre-treatment,7 our method has a 20-fold lower limit of detection

within the same time frame.

One potential drawback of using a random reaction such as

galvanic displacement is that the substrates that form are often highly

variable in morphology and hot spot distribution, yielding a variable

SERS signal. Another potential source for variability in the signal is

the inhomogeneity of the surfaces introduced during themanufacture

and processing or circulation of the tape and coin, respectively. The

variability of the substrates was assessed by calculating a coefficient of

variation (CV) for different points of the same sample droplet, a CV

This journal is ª The Royal Society of Chemistry 2012

for different substrates, and for the Cu coin substrates, a CV for

different points within the same substrate. These CVs are summa-

rized in Table 1. The Cu tape substrates are less variable than the Cu

coin substrates on average, which is not unexpected. The coins come

from circulation and have a high degree of surface heterogeneity,

which is believed to be the main source of variability between

substrates formed on different coins.

Given these sources of variability, we examined the substrates

using scanning electron microscopy to look for similarities since the

limits of detection and average photon count rates were similar

between the two substrates. Fig. 2 shows two scanning electron

micrographs, each revealing a wide variety of fractal, dendritic, and

polygonal Ag structures, consistent with previously published

descriptions of structures formed by galvanic displacement reactions.

It is believed that the large number of branching points and facets

created by the galvanic displacement reaction result in a multitude of

enhancement hotspots distributed across the surface of the substrate.

Furthermore, Supplementary Fig. 1 shows that on the Cu tape, Ag

structures tend to form in a linear arrangement, forming along the

surface striations of the Cu tape.† We then compared the perfor-

mance of our inexpensive and portable substrates with Klarite,

a commercially available SERS substrate. Detection of melamine

extracted and purified from food samples has previously been shown

using Klarite,3,5 so we sought to ascertain its performance without

any sample preparation. Fig. 3 shows spectra from normal infant

formula (0 ppm) and infant formula adulterated with 1000 ppm and

100 ppm melamine using experimental conditions identical to those

responsible for Fig. 1. The 680 cm�1 peak can be seen readily at the

1000 ppm level, but is not apparent at 100 ppm.While the integrated

peak area on the Klarite substrate has a lower CV (0.34) than the

substrates formed by galvanic displacement, it cannot be used under

the same conditions to detect melamine contamination of infant

formula. Thus, these simple, inexpensive, and portable substrates

reported here show up to 200-fold better detection of melamine

contamination in infant formula without pre-treatment than do the

more expensive, commercially available Klarite substrates.

While this direct detection method in its present form does not

approach the sensitivity and specificity of the gold standard methods

such as LC-MS and cannot be used for a quantitative measure of

melamine contamination, the abundant SERS hot spots created by

the galvanic displacement reaction make this a useful method to

rapidly screen batches of infant formula formelamine contamination.

Samples that clearly exceed the limits set by governing bodies can be

quickly pulled from production or distribution, while other samples

Analyst, 2012, 137, 826–828 | 827

Fig. 2 Scanning electron micrographs of representative fractal,

dendritic, and polygonal Ag structures formed on Cu tape (a) and a Cu

coin (b) by galvanic displacement.

Fig. 3 Average SERS spectra of melamine adulterated commercially

available infant formula on Klarite, a commercially available SERS

substrate. Note that the 680 cm�1 peak is readily identifiable at the 1000

ppm level but not at the 100 ppm level. Spectra are offset for clarity.

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with questionable melamine content can be examined by the more

traditional methods that are more precise but far slower. Further-

more, these inexpensive and highly portable SERS substrates can be

combined with commercially available portable Raman spectrome-

ters, enabling SERS analysis to take place in the field or at a point-of

sampling to better detect labile analytes that might not normally

survive the trip back to the laboratory.

Conclusions

Melamine contamination of infant formula can be detected down to

the 5 ppm level without the need for upstream extraction, separation,

purification, or additional equipment using a simple, inexpensive, and

portable SERS substrate formed by the galvanic displacement of Cu

by Ag. Using substrates formed on common, inexpensive, and highly

portable surfaces such as metal tape or coins, contaminated infant

formula samples can be detected in less than 15 min. The fact that

these substrates can be formed in five minutes on-site without

the need for ultra-high vacuum equipment, heating elements,

centrifuges, or harsh chemicals enables the possibility of remote

828 | Analyst, 2012, 137, 826–828

point-of-sampling SERS analysis using commercially available

portable Raman spectrometers and diode lasers.

Acknowledgements

The authors thank Joshua Betz for assistance with statistical analysis.

This research was supported by the Robert W. Deutsch Foundation

and the NSF-EFRI grant NSFSC03524414. The authors acknowl-

edge the support of theMaryland NanoCenter and its NispLab. The

NispLab is supported in part by the NSF as a MRSEC Shared

Experimental Facility.

Notes and references

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This journal is ª The Royal Society of Chemistry 2012