Raman Spectroscopy and Surface Enhanced Raman Spectroscopy

Raman scattering is the inelastic scattering of photons off of molecular bonds. The Raman scattered photons differ in frequency from the incident and elas-tically scattered photons by the frequency of the molecular bond vibration. Figure 13.7 shows an energy level diagram illustrating the Raman scattering process. Most of the incident light is elastically scattered (Rayleigh scattered); however, a small fraction of the incident light (~1 in 108 photons) is Raman scattered. Although the Raman scattering effect was first described in 1928, it did not find wide application until the development of lasers in the 1960s.23-25 The laser provides an intense, monochromatic light source that is ideal for

Elastic Inelastic

Rayleigh Scattering Raman Scattering

FIGURE 13.7 Energy level diagram illustrating the Raman scattering process. Most of the light incident on a chemical bond is elastically scattered, resulting in no change in the frequency of the photon. However, a small fraction of photons are inelastically, or Raman scattered off the molecular bond vibration. In this case the molecule either returns to a higher vibrational energy level, yielding a Stokes shifted photon, or it returns to a lower vibrational energy level, leaving an Anti-Stokes shifted photon.

Elastic Inelastic

Rayleigh Scattering Raman Scattering

FIGURE 13.7 Energy level diagram illustrating the Raman scattering process. Most of the light incident on a chemical bond is elastically scattered, resulting in no change in the frequency of the photon. However, a small fraction of photons are inelastically, or Raman scattered off the molecular bond vibration. In this case the molecule either returns to a higher vibrational energy level, yielding a Stokes shifted photon, or it returns to a lower vibrational energy level, leaving an Anti-Stokes shifted photon.

Raman spectroscopy. Since the development of lasers, Raman spectroscopy has become an important analytical tool for a wide range of disciplines.

Raman spectroscopy provides a wealth of information that can be used to both identify and quantify the molecular species under study. Chemical bonds have unique vibrational frequencies; therefore different molecules have distinct Raman spectra. By comparing the spectroscopic peak positions and relative intensities to those available in a vast library of Raman spectra, molecules can be identified and quantified. Moreover, the information acquired with Raman spectroscopy is complimentary to infrared (IR) absorption spectros-copy, which also measures the frequency of bond vibrations. This provides an additional database for comparing Raman spectra and identifying a particular Raman transition.

Although Raman spectroscopy has gained wide acceptance for analytical chemical applications, its use in biological studies has been somewhat more limited. One reason for the lack of application to biology is the complexity of the signals acquired from biological materials. Biological molecules are typically large, and since the number of vibrational modes is 3n — 6, where n is the number of atoms in the molecule, the spectra can become too congested to interpret. Another reason for the limited application of Raman spectroscopy to biological studies is the intrinsic autofluorescence that can overwhelm the Raman signal. While the Raman spectra for biological samples remain complex, recent advances in near-IR detectors have allowed Raman spectra to be collected with near-IR lasers, thereby reducing the autofluorescence background. As a result, Raman spectroscopy is becoming a more common tool for investigating biological systems.

Raman spectroscopy is particularly useful for identifying conformational states of biological molecules.26,27 It has been applied to study the conformation of lipids in cell membranes, DNA, and proteins. Studies on proteins, for example, have revealed that the amide stretching frequencies in the polypep-tide backbone can be used to identify the secondary structure present in the molecule.26,27 In the Raman spectra of proteins, typically only the amide I and amide III bands are observable. The amide I peak is around 1650 cm-1 for a-helices and shifts to near 1675 cm-1 for b-sheet conformations. Similarly, the amide III peak ranges from 1225 cm-1 to 1245 cm-1 depending on the secondary structure of the proteins. Additionally, other spectroscopic signatures, such as S-S vibrations present in cysteine cross-linking as well as the peaks from aromatic amino acids, help to determine the secondary structure and identity of the protein.

As the library of spectroscopic signatures of biological molecules becomes larger, Raman spectroscopy is beginning to find applications in the biomedical field. Early cancer detection as well as the detection of precancerous cellular changes, the characterization of atherosclerotic plaques, and the identification of pathogenic organisms have all been identified as potential clinical applications for Raman spectroscopy.28,29 Along these lines, bacteria and their endospores have been studied using Raman spectroscopy.30 Early work applying Raman spectroscopy to lyophilized bacterial spores revealed that the primary spectral features observed were due to the calcium dipicolinate present in the cortex of the spore.31 Later work using resonance-Raman spectroscopy to study bacterial spores showed significant differences between the Raman spectra of dormant spores and vegetative bacterial cells.32 This difference was attributed to the release of calcium dipicolinate upon germination. Although other spectral features are observed, such as the amide bands corresponding to the secondary structure of the proteins incorporated in the spore, as well as transitions from some aromatic amino acids, these structural features do not change significantly upon spore germination.

In the present study, we extended the characterization of bacterial spores to the single-spore level. A customized micro-Raman setup (see Fig. 13.8a for a description) allows the sample to be scanned and Raman spectra acquired with spatial resolution in the xy plane on the order of 1 |lm. Samples are then diluted and prepared on a solid substrate such that individual spores are spatially isolated. Figure 13.8b shows a 20 |lm X 20 |lm area of a sample containing Bacillus spores dried onto a calcium fluoride substrate. The bright spots in the image are the fluorescence from individual Bacillus spores. In order to collect the Raman spectra, the spore is centered on the focused laser beam and photobleached for approximately one minute. Once the background fluorescence is reduced in this fashion, the Raman spectra can be collected in as little as 20 seconds.33 The Raman spectra for four different species of Bacillus spores are shown in Figure 13.9. What is striking about the spectra shown in Figure 13.9 is the similarity between the different spore species. The nearly identical spectra are the result of the contribution from calcium dipi-colinate. Calcium dipicolinate is present in the cortex of the spore and comprises ~10% of the dry weight of the spore. While other spectral features that could possibly provide a means of distinguishing between spore species are also clearly present, they are overshadowed by the strong calcium dipicolinate signal.

In order to circumvent this problem, we have employed SERS. SERS is a process through which the Raman signal is greatly enhanced when the chemical species of interest is chemisorbed to a metal (typically gold or silver) surface.34 When the metal surface is illuminated with the appropriate laser wavelength, a surface plasmon resonance is excited. When the surface plas-mons encounter sharp discontinuities in the metal surface, the electric field leaks from the surface at the point of the discontinuity and decays exponentially with distance from the surface, similar to evanescent waves in total internal reflection spectroscopy. This has the effect of focusing the electric field and a)

0 0

Post a comment