RamanStation 400: a Versatile Platform for SERS Analysis
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1 FIELD APPLICATION REPORT Raman Spectroscopy Author: Dean H. Brown PerkinElmer, Inc. Shelton, CT USA RamanStation 400 RamanStation 400: a Versatile Platform for SERS Analysis Introduction Surface Enhanced Raman Spectroscopy (SERS) is a form of Raman spectroscopy which involves the study of samples adsorbed to or interacting in some manner with metal surfaces, typically nanoscale featured gold or silver surfaces, or gold or silver colloids. One limitation of Raman spectroscopy is sensitivity. Only about 0.001% of the incident light produces inelastic Raman signal. SERS offers a solution to this limitation and can even provide a mechanism to inhance sensitivity to physiological or environmental levels. Under the correct conditions, SERS may give rise to spectral enhancements of 10-5 or even 10-6 compared to normal Raman scattering. Working at ppb or femtomol concentration is often straightforward and single molecule detection is widely reported. Research is underway to develop biological molecular sensors for a variety of biomedical applications. 1 Analytical challenges in biomedical research are often selectivity in a complex matrix, sensitivity due to the small amount of sample and the low concentration of the analyte. These challenges must be surmounted. Fortunately, individual sensors can be engineered for the detection of specific molecules due to their chemical selectivity. This is where SERS can also enable researchers to overcome the aforementioned obstacles due to its sensitivity and selectivity. Raman spectroscopy is not affected by water and is compatible with biological systems.
2 Versatility Souza, et. al. 1 used Raman spectroscopy in their efforts to develop networks of SERS gold nanoparticles and bacteriophage as biological sensors and cell targeting agents. Figure 1 shows the Raman SERS spectra of the components in their system. The Raman SERS associated peaks were identified and are shown in Table Well Plate Reader One of the common vehicles used to measure SERS is the use of 96 well plates. The RamanStation 400 can be equipped with a 96 well plate reader. Readings can be taken with or without well caps. Figure 1. Spectra of SERS components used in the development of biological sensors. Table 1. Assignment of SERS bands and amino acids in the spectra shown in Figure 1 (Adapted from 1). Au phage, cm -1 Au phage imid, cm -1 Au imid, cm -1 Imid, cm -1 Side chain or Vibrational imid designation assignmant Imid γ ring 743 cm Imid γ ring 832 cm Tyr, Ile, and/or Met CC 874 cm Imid γ (NH) + δ ring 950 cm -1 1,030 1,030 Tyr, Phe, and/or Met γ (CH) 1,033 cm -1 1,109 1,109 Imid γ (CH) + ν ring 1,097 cm -1 1,169 1,169 1,160 Imid ν ring 1,164 cm +1 1,268 1,268 1,263 Imid δ (CH) 1,265 cm -1 1,328 1,328 Imid ν ring 1,329 cm -1 * δ, in-plane bending; γ, out-of-plane bending; ν, stretching. Figure 2. Auto-subtraction of cap with 96 well plate on an analysis of the anti-inflammatory drug naproxen. 2
3 Automatic Spectral Subtraction Sometimes, the spectral peaks of the plastic from the 96 well plate can contribute spectral peaks to the analysis spectrum. The PerkinElmer RamanStation 400 has the ability to automatically remove the spectra contribution due to the container. The PerkinElmer software offers automatic spectral subtraction using a technique known as the Dwiggle technique, initially described by Li and Banerjee in ,2 The Dwiggle technique does exactly the same thing as the manual subtraction technique, but is fully automated and non-subjective. The RamanStation 400 benchtop Raman spectrophotometer can automatically subtract a reference spectrum in real time to produce a pure spectrum of the sample. This is demonstrated in Figure 3. Figure 3. Spectrum of SERS with and without 96 well plate spectrum removed. An expanded view of the SERS spectrum shows the SERS related peaks (Figure 4). Conformation of SERS peaks is depicted in Table 1. Figure 4. SERS related peaks after spectral subtraction. 3
4 Raman Imaging The X, Y, Z stage increases the versatility of the RamanStation 400 as it allows one to do Raman imaging on biological gels. A visible image is acquired. Then the area to be scanned is selected. Next, the number of data points and spacing of those points are selected. The image is then collected. In Raman chemical imaging, the spectra are acquired in a grid pattern (or map) and processed using software to provide a two-dimensional representation of the sample composition. This map can be constructed from any number of spectral properties such as the intensities or areas of particular peaks of interest, but importantly, these spectral properties can be chosen to reflect the chemical composition. Resolution Peak resolution 4 cm -1 FWHM, (measured using the calcite band full width half maximum) pixel resolution 0.75 cm -1 with linear cm -1 dispersion across entire spectral range. Full spectral range can be acquired at this resolution in a single acquisition. High resolution spectra are important for spectral separation for both qualitative and quantitative analysis. Figure 6. High resolution spectrum from RamanStation 400. Autobaseline Correction PerkinElmer s software incorporates a technique which fits a high order polynomial curve to the data. The software then analyzes the original spectrum and the polynomial, and generates a new spectrum (Figure 7) which is made of the original spectrum, with all data-points more intense than the polynomial removed. This resulting spectrum is the software s first approximation at the fluorescent baseline. This polynomial fitting is repeated, this time using the first pass baseline as the starting point. This process is repeated up to 30 times, resulting in a polynomial which hugs the baseline of the original spectrum. Subtraction of the final polynomial from the original spectrum yields the baseline corrected spectrum. Figure 5. Visual image of sample, Raman image and extracted spectrum from image. Figure 7. Automatic baseline collection on the RamanStation
5 Validation Since there are no moving parts in an Eschelle grating, it can be calibrated and trusted. Validation using a NIST 2241 intensity standard insures consistent and validated spectra. This is important when comparing spectra for qualitative verification and for publication and inter-laboratory comparisons. Auto Exposure Time Calculation Trying to determine the optimum combination of number of scans and scan-times for each type of sample can be difficult to do for the inexperienced Raman user. The RamanStation 400 has two modes of data collection. For those analysts who know the best scanning conditions for a particular sample or who want to compare spectra collected under the same conditions, there is a manual option to specify the number and duration of the scans. There is also an automatic mode where the analyst simply enters the total accumulation time for the analysis and the system will calculate the best combination of number of scans and scan-times to give the optimum spectrum. It does this by opening the laser shutter and monitoring the signal reaching the detector for a short time. This function takes into account the intensity of the Raman signal, detector saturation, and background fluorescence. There will always be at least two scans collected in order to minimize any unwanted effect caused by cosmic rays. Variable Spot Size The RamanStation 400 provides a variety of sampling modes that increase the versatility and enable the system to acquire the best data possible. Single point mode The sample is measured at a single point. SuperMacro point mode Measures a center and three points on either side of the central point for a total of 7 analysis spots. This could be helpful sampling a larger area for a more homogeneous sampling area. Figure 8. Calibrated and uncalibrated spectra compared to the NIST 2241 standard. Variable Laser Power Control Analysis of samples containing dark pigments or carbon black may lead to complications in analyzing by Raman spectroscopy. This is due to absorption of the incident laser energy and the emitted Raman scatter by the sample. This can lead to burning or deformation of the sample. The sample absorption reduces the Raman intensity thus producing noisy spectrum. This does not prohibit the analysis of these types of samples but steps will need to be taken to prevent the degradation and to increase the signal to noise of the sample spectrum. 5
6 One of the obstacles that must be overcome in Raman spectroscopy is the possibility that the laser will destroy the sample thus preventing analysis. One parameter that can be varied to prevent the destruction of the sample is to reduce the power of the laser. Figure 9 shows a polymer sample before and after analysis. The sample has been pitted and photo degraded. Figure 9. Photo degradation of sample from the laser. The RamanStation 400 allows the analyst to control the power of the laser. The power can be set from 5 to 100%. Flexible Sample Handling A full range of clip-in sampling accessories and drop-in sample holders ensures the widest range of sample types can be accommodated. The modular sample compartment makes it ideal for running everything from routine samples in glass vials to automated quantitative analysis of multiple samples on well-plates. Microscopes and fiber probes can also be fitted to the external sampling port. Interchangeable Sampling Modules Standard Sample Holder RamanStation 400 s softwarecontrolled X, Y, Z sample stage automatically aligns your sample in all three coordinates to obtain the optimum spectrum. Large numbers of spectra can be acquired quickly, providing reproducible, research-quality results all at the touch of a button. A Windows -based Video in Window Camera The 30 X magnification video camera image facilitates precise sample alignment. Visible Image Survey allows image collection for samples larger than the camera field of view. Conclusion The RamanStation 400 is a versatile Raman spectrometer that is flexible for SERS analysis. It has a 96 well plate reader and is capable of collecting Raman images. The automatic baseline subtraction, spectral subtraction, and auto exposure calculation simplifies the acquisition of high quality SERS data with minimal interferences. The variable spot size, variable laser power capabilities, and interchangeable accessories make the RamanStation 400 flexible enough to analyze almost any sample. Finally, the ability to validate the instrument and produce high resolution spectra makes it ideal for publication and inter-laboratory comparisons. References 1. Souza, G.R., et. al., (2006) Proceedings of the National Academy of Sciences, 103, K. Li and S. Banerjee, Appl. Spec., Vol. 45, 1991, p M.A. Friese and S. Banerjee, Appl. Spec., Vol. 46, 1992, p The compact sample enclosure offers various sample holding inserts to accommodate cuvettes, vials, small bottles and powder cups. Stainless steel powder cups are provided to hold neat powders and films. With the sample holder tipped on its end, solids can be measured directly to avoid any spectral contribution from glass containers. PerkinElmer, Inc. 940 Winter Street Waltham, MA USA P: (800) or (+1) For a complete listing of our global offices, visit Copyright 2012, PerkinElmer, Inc. All rights reserved. PerkinElmer is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners _01
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