Infection Detection: Raman Spectroscopy's Role in C. Difficile Testing

Raman spectroscopy is an analytical method of chemical identification that relies on the inelastic scattering of photons from a laser through the sample in question. This analytical technique is also applied to biological molecules, such as those associated with bacterial infection, and thus can be exploited in infection diagnosis.

Image Credit: ART-ur/

Image Credit: ART-ur/

This article will discuss Raman spectroscopy and some of its sub-types, with a view to their specific application towards the detection of C. Difficile infection.

Decoding Raman Spectroscopy: A Light-Based Analytical Technique

Raman spectroscopy depends on the inelastic scattering, or Raman scattering, of photons from the molecule and is based on the interaction of monochromatic light with the electron cloud of the sample. The external electric field of incident monochromatic light can induce a dipole moment within the molecule based on its polarizability. Thus, the intensity of Raman shift is proportional to the degree of polarizability change.

There are numerous specific types of Raman spectroscopy that exploit a range of optical and electromagnetic phenomena for analytical purposes, and each operates by somewhat differing principles. For example, in ordinary Raman spectroscopy, the incident laser light causes a shift in the vibrational state of the molecule, dependent on the existing atoms and bonds between them from which it is constructed, and does not involve the promotion of electrons to higher energy levels and subsequent emission.

On the other hand, X-ray Raman spectroscopy relies on higher energy X-rays to induce the promotion of inner shell electrons and subsequent photon emission upon degradation, allowing the identification of constituent atoms and the probable bonds between them. In any case, Raman spectroscopy provides a "fingerprint" of the analyzed molecule, measured in the intensity of Raman shift per wavenumber in cm-1.

Revolutionizing Infection Diagnostics with Raman Spectroscopy

The molecular fingerprinting potential of Raman spectroscopy extends to large biological molecules such as proteins, including those produced by viruses and bacteria. Thus, Raman spectroscopy is a useful tool in infection diagnostics. Raman spectroscopy is typically a laboratory technique where specific preparatory steps are taken, depending on the sample, to obtain an optimally true measurement of molecule properties.

Ordinary swabs, saliva, or blood samples can be examined using Raman spectroscopy for the presence of pathogens, but at this time, they are generally prepared to reduce unwanted detection of much more concentrated biomolecules. Hand-held Raman spectrometry machines have been produced but are generally intended to identify mostly pure products such as drugs.  

Unveiling C. Difficile Infections

C. Difficile infections are common in hospital settings and account for a large proportion of antibiotic-associated diarrhea, antibiotic-associated colitis, and almost all cases of antibiotic-associated pseudomembranous colitis. Infection is typically confirmed by testing stool samples for molecules associated with bacterial waste, Cell Culture Cytotoxicity Neutralization Assay (CCNA) being considered the gold standard.

Typically, this assay contains human fibroblast cells that react in a specific manner to the presence of toxins, though this method is labor intensive and requires at least 48 hours of incubation. Other less specific techniques than CCNA are available in the identification of C. Difficile, such as enzyme immunoassay or toxigenic culture and nucleic acid amplification testing (NAAT), but these techniques can also be costly and may fail to differentiate between toxigenic and non-toxigenic strains.

Raman spectroscopy has recently been explored in the fast and efficient identification of toxigenic C. Difficile infections via stool samples, specifically by recognition of the fingerprints of two toxins, showing comparable sensitivity and specificity to CCNA, with a much quicker turnaround time.

Advancements in Sensitivity and Specificity

Another form of Raman spectroscopy not yet discussed is surface-enhanced Raman spectroscopy (SERS), which significantly enhances Raman scattering when molecules are absorbed onto nanostructured surfaces, often by ten or more orders of magnitude, even allowing the detection of single molecules. This is likely due to the influence of localized surface plasmon resonance surrounding nanostructured objects constructed from plasmonic materials such as gold or silver.

Typically, roughened flat surfaces can produce nanostructured planes capable of SERS, but this effect can also be produced by molecular proximity to plasmonic metal nanoparticles. The surface of these nanostructured materials can be functionalized with molecules complementary to the desired target molecule, such as an antibody specific to a bacterial antigen.

In this way, a mixture of biomolecules in a recently collected sample can be mixed with functionalized nanoparticles that will bond with only the target molecule and then either analyzed by SERS directly, the greatly amplified signal drowning out unwanted noise or alternatively can be easily separated by centrifugation owing to the much higher density of noble metal nanoparticles than anything else in biological solution.

Beyond the Lab: Potential Clinical Integration

Raman spectroscopy is a rapid, cost-effective, and efficient method of analysis, and compared to similarly convenient and accurate analytical techniques can be performed in a non-contact manner with the sample, owing to the use of laser light, making it suitable for the testing of stool samples. Combined with carefully engineered nanotechnological innovations, Raman spectroscopy can detect extremely low concentrations of target analyte, potentially even within complex biological mixtures.

Given the prevalence of C. Difficile infections within hospital settings and the high prevalence of over-diagnosis of the infection, fast and convenient methods of diagnosis are called for. The higher initial cost of Raman spectroscopy equipment may slow the initial adoption for the sole purpose of C. Difficile identification, while the use of specific SERS platforms may prove costly in the short term.


In conclusion, Raman spectroscopy is a powerful and rapid analytical technique that allows the highly specific identification of molecules based on how they inelastically scatter light. There is a wide variety of specific types of Raman spectroscopy intended for particular applications, but in its most generic form, it can be useful in the identification of pathogenic infections.

The technique may see more use in a clinical setting as it becomes increasingly cost-effective and broadly available and as specific techniques in identifying certain infections are further developed.


  • Koya, Satya Kiran; Yurgelevic, Sally; Brusatori, Michelle; Huang, Changhe; Diebel, Lawrence N.; Auner, Gregory W. (2019). Rapid Detection of Clostridium difficile Toxins in Stool by Raman Spectroscopy. Journal of Surgical Research, 244(), 111–116.
  • Lukose, J., Barik, A. K., Mithun N, Sanoop Pavithran M, George, S. D., Murukeshan, V. M., & Chidangil, S. (2023). Raman spectroscopy for viral diagnostics. Biophysical Reviews15(2), 199–221.
  • Jones, R. R., Hooper, D. C., Zhang, L., Wolverson, D., & Valev, V. K. (2019). Raman Techniques: Fundamentals and Frontiers. Nanoscale Research Letters14(1).

Further Reading

Last Updated: Oct 17, 2023

Michael Greenwood

Written by

Michael Greenwood

Michael graduated from the University of Salford with a Ph.D. in Biochemistry in 2023, and has keen research interests towards nanotechnology and its application to biological systems. Michael has written on a wide range of science communication and news topics within the life sciences and related fields since 2019, and engages extensively with current developments in journal publications.  


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