The Use of Raman Spectroscopy in Cancer Diagnostics

Sep 01, 2013
Volume 28, Issue 9

Both Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) are proving to be invaluable tools in the field of biomedical research and clinical diagnostics. The robust, compact, fit-for-purpose Raman spectrometer designs are appropriate for use in surgical procedures to help surgeons assess tumors and allow rapid decisions to be made. Raman systems are also being developed for molecular diagnostic testing to detect and measure human cancer biomarkers. Based on the SERS technique, this approach potentially could change the way bioassays are performed to improve both the sensitivity and reliability of testing. The two applications highlighted in this review, together with other examples of the use of Raman spectrometry in biomedical research areas such as the identification of bacterial infections, are clearly going to make the technique an important part of the medical toolbox, as we continually strive to improve diagnostic techniques and bring a better health care system to patients.

In recent years, Raman spectroscopy has gained widespread acceptance in applications that span from the rapid identification of unknown components to detailed characterization of materials and biological samples. The technique's breadth of application is too wide to reference here, but examples include quality control (QC) testing and verification of high purity chemicals and raw materials in the pharmaceuticals and food industries (1–3), investigation of counterfeit drugs (4), classification of polymers and plastics (5), characterization of tumors, and the detection of molecular biomarkers in disease diagnostics (6–8) and theranostics (9).

One of the most exciting application areas is in the biomedical sciences. The major reason behind this surge of interest is that Raman spectroscopy is an ideal technique for molecular fingerprinting and is sensitive to the chemical changes associated with disease. Furthermore, components in the tissue matrix, principally those associated with water and bodily fluids, give a weak Raman response, thereby improving sensitivity for those changes associated with a diseased state (10). The growing body of knowledge in our understanding of the science of disease diagnostics and improvements in the technology has led to the development of fit-for-purpose Raman systems designed for use in surgical theaters and doctors' surgeries, without the need for sending samples to the pathology laboratory (8).

Surface-enhanced Raman spectroscopy (SERS) is a more sensitive version of Raman spectroscopy, relying on the principle that Raman scattering is enhanced by several orders of magnitude when the sample is deposited onto a roughened metallic surface. The benefit of SERS for these types of applications is that it provides well-defined, distinct spectral information, enabling characterization of various states of a disease to be detected at much lower levels. Some of the many applications of Raman and SERS for biomedical monitoring include
  • Examination of biopsy samples
  • In vitro diagnostics
  • Cytology investigations at the cellular level
  • Bioassay measurements
  • Histopathology using microscopy
  • Direct investigation of cancerous tissues
  • Surgical targets and treatment monitoring
  • Deep tissue studies
  • Drug efficacy studies

The basics of Raman spectroscopy are well covered elsewhere in the literature (11). However, before we present some typical examples of both Raman and SERS applications in the field of cancer diagnostics, let's take a closer look at the fundamental principles and advantages of SERS.

Principles of Surface-Enhanced Raman Spectroscopy

The principles of SERS are based on amplifying the Raman scattering using metal surfaces (usually Ag, Au, or Cu), which have a nanoscale roughness with features of dimensions 20–300 nm. The observed enhancement of up to 107-fold can be attributed to both chemical and electromagnetic effects. The chemical component is based on the formation of a charge-transfer state between the metal and the adsorbed scattering component in the sample and contributes about three orders of magnitude to the overall enhancement. The remainder of the signal improvement is generated by an electromagnetic effect from the collective oscillation of excited electrons that results when a metal is irradiated with light. This process, known as the surface plasmon resonance (SPR) effect, has a wavelength dependence related to the roughness and atomic structure of the metallic surfaces and the size and shape of the nanoparticles. For a more detailed discussion of SERS and its applications, see reference 12.

Detection by SERS has several benefits. Firstly, fluorescence is inherently quenched for an adsorbate analyte on a SERS surface resulting in fluorescence-free SERS spectra. This is primarily because the Raman signal is enhanced by the metal surface, and the fluorescence signal is not. As a result, the relative intensity of the fluorescence is significantly lower than the Raman signal and in many occasions is not observable. Secondly, signal averaging can be extended to increase sensitivity and lower detection levels. Finally, SERS spectral bands are very sharp and well-defined, which improves data quality, interpretability, and masking by interferents. Recent work using silver nanoparticles as the enhancing substrate has shown that SERS can be applied to single-molecule detection, rivaling the performance of fluorescence measurements (13).

Although SERS has many advantages it also has several disadvantages that are worth noting. The first is that unlike traditional Raman spectroscopy, SERS requires sample preparation. The ability to measure samples in vivo without the need for sample pretreatment is one of the major advantages of traditional Raman spectroscopy. For that reason, it is important to understand the signal enhancement benefits of SERS compared to the ease of sampling of traditional Raman spectroscopy when deciding which technique to use (14). Second, high performance SERS substrates are difficult to manufacture and, as a result, there is typically a high degree of spatial nonuniformity with respect to the signal intensity (15). For example, the SERS substrates used in the research being carried out by the University of Utah (described later in this article) tend to have much higher concentrations of analyte on the edges of the sampling area than in the center. Lastly, it is important to note that when the sample is deposited onto the surface, the molecular structure (the nature of the analyte) can be altered slightly resulting in differences between traditional Raman spectra and SERS spectra (16). However, it should be emphasized that there have been improvements in the quality of substrates being manufactured today and new materials such as Klarite (Renishaw Diagnostics) are showing a great deal of promise in terms of consistency and performance (17).


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