Melamine is a dangerous substance when it is used to increase the apparent protein content of food. In this article, the use
of novel gold nanoparticles for surface-enhanced Raman spectroscopy (SERS) measurements with raster orbital scanning sampling
is described for the detection of melamine and other food contaminants. Although silver nanoparticles are often used for melamine
SERS measurements, gold nanoparticles are more stable with less stringent requirements for storage and handling. Gold SERS
substrates can also be used for the detection of other harmful food additives and adulterants, including antifungal dyes,
antibiotics, and pesticides.
Food safety is a global concern that has spawned rigorous guidelines and numerous regulatory agencies to protect consumers
from both harm and fraud (1). Widespread illness and even death have resulted from the adulteration of food with compounds
like melamine, which are added to ensure substandard foods meet dietary guidelines. Trace contaminants and residues from pesticides
and antifungal agents used in the growth, processing, and storage of food products have been shown to be toxic to humans with
impacts ranging from digestive problems to death. Counterfeit food products deceive consumers, risking their health while
emptying their wallets. It is these ongoing deceptive and dangerous practices and the widespread illness and economic impact
they have caused that have driven the establishment of strict food safety regulations and the need for careful food inspection.
Consumers rely on government scrutiny and oversight for protection from dangerous food additives and contaminants. The use
of melamine and its derivative, cyanuric acid, have invited scrutiny and inspired tighter regulation of fungicides, pesticides,
and antibiotics used for crop protection. The antifungal agents crystal violet and malachite green are inexpensive and effective
against fungal and parasite infections in fish, but are not approved for use in aquaculture because of their mutagenic impact
on humans. The fungicide thiram is used to protect crops from deterioration both before and after harvest, but has high enough
toxicity that it requires protective clothing for handling. The broad-spectrum antibiotic chloramphenicol is banned for food
use and is tightly regulated to avoid toxicity and the potential for the creation of antibiotic-resistant bacteria. These
compounds and many others have documented health and safety concerns requiring regulation and inspection of food products.
With the potential for serious health and economic effects, there is a need for a rapid, low cost, and sensitive technique
to detect and identify harmful food contaminants. Although there are a variety of methods and techniques now available for
the identification and quantification of these potentially harmful compounds, these techniques (high performance liquid chromatography–mass
spectrometry [HPLC–MS], gas chromatography–mass spectrometry [GC–MS], and ion-mobility spectrometry [IMS]) are often expensive,
time- and labor-intensive measurements requiring significant sample preparation and highly trained personnel to administer.
For a viable, commercial application, a method is needed that requires little to no sample preparation, is applicable to a
range of food contaminants, and can provide fast, accurate identification and quantification. Surface-enhanced Raman spectroscopy
(SERS) meets these requirements and is a good option for the detection of food contaminants and additives.
Raman spectroscopy is a high-resolution, vibrational spectroscopic technique in which each molecule produces a unique spectral
"fingerprint" that can be used to identify and differentiate it from other sample components. Recent advances in lasers, detectors,
and optical filter technologies have lowered costs and enabled considerable downsizing of Raman instrumentation such that
the technology is more accessible and field deployment is now feasible. As a detection technology, Raman spectroscopy (2)
has gained increasing interest because the Raman band frequencies relate to chemical bonding in the compound to be identified.
This chemical specificity gives detection by Raman spectroscopy a distinct advantage. Different compounds have unique Raman
fingerprints that can be used to identify them.
The major drawback of Raman spectroscopy is the low cross-section (2) of the spontaneous Raman scattering, which affects the
detection of substances at trace level. This small scattering cross-section results in lower Raman signals and makes detection
and identification difficult for compounds like food contaminants and additives that are present at low concentrations. In
1977, a technique to overcome this limitation was demonstrated when the compound of interest was placed on a roughened noble-metal
substrate. The result was a significant enhancement in the magnitude of the Raman scattering signal (3). This process is known
as surface-enhanced Raman scattering (4,5).
The enhanced sensitivity achievable with SERS makes it a viable option for the detection of food contaminants often present
at trace levels. SERS is a variation of conventional Raman spectroscopy whereby analytes are adsorbed onto a noble metal (typically
gold or silver) covered surface before analysis. Through a combination of chemical and electromagnetic effects, the Raman
signal intensity is significantly "enhanced" when measured from SERS substrates fabricated from these noble metals. In very
specific cases, detection at the single-molecule level has even been demonstrated (6,7). The combination of signal enhancement
and chemical specificity make SERS a good candidate for the sensitive and selective identification of compounds including
harmful food contaminants and additives (8,9).
Since the early days of SERS, gold (Au) and silver (Ag) have been widely used as SERS substrates because of their strong SERS
activity (10). The use of these metals has advanced with the development of nanoscience and nanotechnology, which give researchers
the ability to control the shape, size, and composition of silver and gold nanoparticles. As a result, silver and gold are
the most widely used SERS substrates. Theoretical calculations indicate that the Raman enhancement of a single gold nanoparticle
is about 103 –104 with enhancement for a single silver nanoparticle as high as 106 –107 (11).
In this paper, we describe the novel combination of gold substrates and raster orbital scanning (ROS) for the detection of
melamine and other harmful food additives and contaminants. Data illustrating the increased stability of the gold substrates
relative to silver substrates are shown along with the enhancement in signal achieved with ROS sampling versus traditional
Raman sampling. The broad applicability of the gold SERS substrates is also shown for several other food contaminants.
Although melamine is typically detected using silver SERS substrates with 532-nm laser excitation, we used gold substrates
with 785-nm laser excitation for our measurements. Although melamine binds well to silver, one of the limitations of the silver
substrates is a shorter lifetime, requiring modified atmospheric packing of the substrates and special storage and handling
conditions (12,13). Gold, on the other hand, ages much more slowly with better stability than silver (14,15). This increased
stability and longer lifetime are why we used gold substrates even though melamine binds better to silver. Gold also has the
added benefit of working with a broader range of harmful food additives and contaminants.
When the more stable gold SERS substrates are combined with an ROS Raman sampling technique, the result is a rapid, sensitive
method for detecting several regulated or banned food additives and contaminants. ROS combines the resolution and power achieved
with a tightly focused laser spot with the sensitivity of sampling over a large sample area. By rastering the laser spot over
a large sample area, there is a higher probability of measuring spectra from SERS hotspots (localized regions of Raman signal
enhancement) dispersed on the substrate. This results in higher sensitivity than with traditional Raman sampling where the
laser is focused on a single location on the substrate. This is especially important with SERS substrates, in which the density
and coverage of nanoparticles on the substrate can vary from location to location. Not only does ROS overcome this challenge
of working with SERS substrates, it also provides a lower average laser power at the substrate and reduces the possibility
for substrate or sample damage during exposure to the tightly focused laser.