Detection of Harmful Food Contaminants Using SERS
Written by Ocean Optics Staff
Melamine is a dangerous substance when used to increase the apparent protein content of food. Widespread illness and several deaths are attributed to the kidney toxicity associated with melamine ingestion. Health scares resulting from melamine adulteration in both human and pet food have driven the search for a reliable and sensitive technique for its detection. With high specificity and sensitivity, Surface-Enhanced Raman Scattering (SERS) offers a viable alternative to chromatographic and other screening techniques now used for melamine testing. When SERS is combined with a Raster Orbital Scanning (ROS) sampling technique, the larger sample area interrogated by the laser improves sensitivity with a lower average laser power that reduces the risk for substrate or sample damage.
In this paper, we describe the use of gold nanoparticles for SERS measurements with ROS sampling for the detection of melamine and other food contaminants. While silver nanoparticles are often used for melamine SERS measurements, gold nanoparticles are more stable and have less stringent requirements for storage and handling. Also, gold SERS substrates can 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 spurred numerous regulatory agencies to protect consumers from both harm and fraud (1). Serious health issues have resulted from the adulteration of food with compounds like melamine, which are added to ensure substandard foods meet dietary guidelines. Also, 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 effects ranging from digestive problems to death.
Consumers rely on government 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. For example, the anti-fungal agents crystal violet and malachite green are inexpensive and effective against fungal and parasitic infections in fish but are not approved for use in aquaculture due to 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.
With the potential for serious health and economic consequences, there is need for a rapid, low cost and sensitive technique to detect and identify harmful food contaminants. Although there are methods and techniques now available for the identification and quantification of harmful compounds, these techniques often require expensive, labor-intensive measurements involving 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 detection of food contaminants and additives.
Advantages of Raman Spectroscopy
Recent advances in lasers, detectors, and optical filter technologies have lowered costs and enabled dramatic downsizing of Raman instrumentation, making it considerably more accessible. Raman spectroscopy (2) has gained interest because 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 phenomenon results in lower Raman signals and makes detection and identification difficult for compounds like food contaminants 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 (SERS) (4, 5).
The enhanced sensitivity achievable with SERS makes it a viable option for the detection of food contaminants 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 prior to 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 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 due to their strong SERS activity (10). The use of these metals has advanced with the development of 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 and enhancement for a single silver nanoparticle is as high as 106 -107 (11).
In this paper, we describe how the novel combination of gold substrates and ROS sampling is used for the detection of melamine and other harmful food additives and contaminants. Data showing the increased stability of the gold substrates relative to silver substrates is 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.
Even though 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. While 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). Also, gold has the benefit of working with a broader range of harmful food additives and contaminants.
When 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, where 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.
Silver and gold nanoparticles were inkjet printed on cellulose substrates. For substrate aging studies, melamine was tested on the day of printing and up to one week after printing. Measurements were made by drop-casting 12 μL of the compounds suspended in various solvents to the printed SERS substrates mounted on standard glass microscope slides.
Substrates were interrogated using one of two setups: our IDRaman reader integrated Raman system with 785 nm laser excitation and ROS sampling; and our modular Raman system comprising a QE Pro spectrometer configured for 785 nm Raman measurements, along with a 785 nm laser for excitation and a Raman probe for detection. Typical acquisition parameters were 3 scans to average and a 1 second integration time. Data was acquired using OceanView spectroscopy software.
In all the plots presented, the spectra were adjusted for an appropriate baseline to enable comparison of peak height values. A Raman-inactive area of the spectrum was determined for each analyte, and the average value across that region was subtracted from the rest of the spectrum. This achieves a baseline correction that accounts for the noise seen in the raw spectrum. The spectra reported are raw spectra that have been baseline corrected.
The observed shifts in the assigned peaks for the analytes could be due to a variety of factors. It is known that differences in solvents and, more important, physical features in SERS substrates will cause the observed peaks to change. This explains any variation in peak assignment when compared to reference literature.
Results and Discussion: Impact of Substrate Aging
To demonstrate the impact of substrate aging on the SERS detection of melamine, both silver and gold nanoparticles were inkjet printed on cellulose substrates. Melamine at 1×10-2 M concentration in water was drop-casted to the substrates and Raman spectra were measured on the day of printing and 24 hours after printing.
The Raman spectra measured with the modular Raman system using traditional Raman sampling for melamine on a silver substrate are shown in Figure 1. Note the characteristic peak for melamine near 700 cm-1 in the spectrum measured just after the substrate was printed (Day 0). Deterioration of the silver substrate is observed by the loss of this peak in the Raman spectrum measured 24 hours after the substrate was printed (Day 1). The silver substrate degraded significantly within 24 hours of printing, resulting in an inability to detect melamine.
When a gold substrate is used to measure the same melamine solution, the improvement in stability is dramatic. As shown in Figure 2, the characteristic melamine peak is still observed one week after the gold substrate was printed. Additional studies have demonstrated even longer stability – more than 60 days after printing. This increased stability makes gold substrates easier to work with than silver substrates and more suitable for use in commercial applications.
The improvement in SERS measurements for melamine is even better when the excellent stability of the gold substrates is combined with the ROS sampling technique. When the relative intensity of the Raman peaks for melamine in Figure 2 are compared with the relative intensity of the melamine peaks in Figure 3, the signal measured with ROS sampling (Figure 3) is almost two times the relative intensity of the signal measured with traditional Raman sampling (Figure 2). This increase in Raman intensity measured using ROS sampling demonstrates the dramatic improvement in sensitivity achieved with ROS when a larger area of the SERS substrate is sampled.
Detection of Other Compounds of Food Safety Concern
As demonstrated in Figures 4-7, gold SERS substrates are useful for the detection and discrimination of potentially harmful food contaminants and additives. The Raman spectrum for the fungicide Thiram is shown in Figure 4. The sample was prepared by drop-casting a solution containing 1 x 10-3 M Thiram in acetone to the gold substrate. This toxic fungicide is used to prevent crop damage before and after crop harvest. The toxicity of this fungicide is high enough that it requires protective clothing for handling. As shown in Figure 4, gold SERS substrates and ROS sampling have sufficient sensitivity to detect relatively low levels of this toxic fungicide.
Chloramphenicol is a broad-spectrum antibiotic used in aquaculture. It has been banned for food use and is tightly regulated to avoid toxicity and the potential for the creation of antibiotic-resistant bacteria. As shown in Figure 5, SERS with a gold substrate and ROS sampling enables detection of 1 x 10-3 M concentrations in ethanol of this banned food contaminant.
In Figures 6-7, Raman spectra for the fungicides crystal violet and malachite green are shown. These fungicides are also banned for use in aquaculture due to their toxicity. These anti-fungal agents are low cost and very effective against fungal and parasitic infections in fish, but they are not approved for use in aquaculture due to their potential for mutagenic impact on humans. A comparison of these spectra demonstrates not only the sensitivity of the technique but also the specificity of Raman analysis. These fungicides have distinct spectral fingerprints allowing for discrimination and identification. As shown in Figures 6-7, both fungicides are easily detected at 1 x 10-4 M concentrations in ethanol with SERS using gold substrates and ROS sampling.
Food safety is a global concern. Outbreaks and even death have driven the search for improved technologies to detect very low levels of food contaminants. The tremendous signal enhancement associated with SERS, combined with ROS sampling of highly stable gold substrates, make SERS a good candidate for the detection of trace levels of food additives and contaminants. With recent advances in handheld Raman instrumentation including the availability of handheld devices with ROS sampling, SERS is a viable option for the fast, low cost and sensitive detection of these dangerous compounds outside the laboratory setting.
- K.R. Matthews, in Practical Food Safety: Contemporary Issues and Future Directions, R. Bhat and V. Gomez-Lopez, Ed. (John Wiley and Sons, Hoboken, New Jersey, 2014), pp. 1-9.
- D.J. Gardiner, in Practical Raman Spectroscopy, D.J. Gardiner and P. R. Graves, Ed. (Springer-Verlag, Berlin, 1989), pp. 1-12.
- D.L. Jeanmarie, R.P. Van Duyne, J. Electroanal. Chem. 84, 1–20 (1977).
- M. Fleischmann, P.J. Hendra, and A.J. McQuillan, Chem. Phys. Lett. 26(2), 163–166 (1974).
- R. A. Alvarez-Puebla, D. S. Dos Santos Jr and R. F. Aroca, Analyst, 129, 1251 – 1256 (2004).
- W.E. Doering, S.M. Nie, J. Phys. Chem. B, 106, 311-317 (2002).
- K. Kneipp, H. Kneipp, Appl. Spectrosc., 60(12), 322A-334A (2006).
- A.P. Craig, A. S. Franca, J. Irudayaraj, Ann. Rev. Food Sci. Technol. 4, 369-380 (2013).
- J. Zheng, L. He, Comp. Rev. Food Sci. Food Safety, 13, 317-329 (2014).
- J.A. Creighton, C.G. Blatchford, M.G. Albrecht, J. Chem. Soc. Faraday Trans. 75, 790-798 (1979).
- D.S. Wang, H. Chew, M. Kerker, Appl. Opt. 19, 2256-2257 (1980).
- N.L. Rosi, C.A. Mirkin, Chem. Rev. 105, 1547-1562 (2005).
- M.D. McMahon, R. Lopez, H.M. Meyer III, L.C.R.F. Feldman, J.R. Haglund, Appl. Phys. B 80, 915-921 (2005).
- P. Gao, M.J. Weaver, J. Phys. Chem. 89, 5040-5046 (1985).
- P. Gao, D. Gosztola, L.W.H. Leung, M.J. Weaver, J. Electroanal. Chem. 233, 211-222 (1987).