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The medical industry is a new frontier for Raman spectroscopy. In the past few years, Raman has been used in dental and cancer studies, and is now building on this success by extension into Point-of-Care (POC) applications. This article gives an overview of some very new and exciting research into the use of Raman spectroscopy for the detection of cancerous tissue, biomarkers of disease, and pathogens that cause disease. 

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Detecting bone infection with a handheld Raman spectrometer

Complication from infection is a serious issue for the use of human bone grafts in musculoskeletal surgery. Staphylococcus epidermidis and Staphylococcus aureus are the most common culprits in bone-related infections, and treatment of these bacteria is extremely difficult.

Collage of many X-rays of joints.

Detection of staph bacteria on graft material and distinction between healthy and diseased bone are both crucial in preventing infection. Laboratory culture traditionally requires 7–10 days for results and carries the risk of contamination during transport and testing. In positive cases, the patient must be treated retroactively with massive doses of antibiotics. An ideal solution for this challenge is on-site analysis that permits the surgical team to identify and avoid diseased bone on the spot.

Recently, a research group in Austria demonstrated successful differentiation between healthy and infected bone samples and between two types of staph bacteria using a handheld MIRA Raman spectrometer [1]. Their procedure analyzed the fingerprint Raman bands of phosphates, amides, and collagens, and their differing intensity and peak width ratios to distinguish between healthy and diseased bone. Principal component analysis (PCA) supported optical analysis and was used in the finer distinction of staph strains.

The Austrian group appreciated that MIRA was «lightweight, compact, and battery powered» and that Raman spectroscopy has the benefit of «minimal sample preparation and rapid results.» Testing required only a tiny bone sample for in-situ examination during surgery and provided fast and accurate results directly in the operating room.

Raman spectroscopy for cancer detection

Raman’s «molecular fingerprint» spectra can be sensitive enough to detect the chemical changes that accompany disease. Compact Raman spectrometers can help surgeons assess tumors during surgical procedures for faster decision-making. Application examples for breast cancer and pancreatic cancer are discussed in the following sections.

Traditional Raman spectroscopy in breast cancer assessment

When breast cancer is suspected, one surgery is typically required for biopsy and a second is for removal of malignant tumors. The ability to assess suspect tissues during preliminary surgery permits immediate removal, if required. The benefit to the patient and to the medical industry is inestimable—and research suggests that Raman spectroscopy may be able meet this need for some types of cancer.

Raman spectroscopy is sensitive enough to detect changes in tissue that result from many types of cancer. For example, there are very subtle differences in Raman spectra from healthy breast tissue and malignant tumors. Researchers in the UK used a high-resolution i-Raman laboratory device (Figure 1) and multivariate tools, including PCA, for successful discrimination of healthy and cancerous tissues [2].

Figure 1. The i-Raman Prime 785S Portable Raman Spectrometer from Metrohm.

SERS for detection and measurement of pancreatic cancer biomarkers

SERS (surface-enhanced Raman spectroscopy) can be a solution when Raman is not ideal for analysis. This may be the case in situations where the target exists in a complex sample matrix, or because of the fluorescence of carbon-based molecules. SERS enhances the Raman signal, but not the competing fluorescence signal. Also, the SERS effect enables sensitive detection of analytes at mg/L levels—sometimes down to µg/L. Finally, SERS peaks are sharp and well-defined, allowing efficient detection and identification of target analytes.
 

To learn more about SERS, read our previous blog post on this topic.

Raman vs SERS… What’s the Difference?


Pancreatic cancer is deadly, partly because it is hard to diagnose. However, there are some biomarkers that are present at elevated levels in ~75% of positive cases [3]. These can be detected with enzyme-linked immunosorbent assays (ELISA), which measure a variety of biomarkers, including antibodies, antigens, and proteins.

In an emerging technique, an i-Raman laboratory spectrometer was used at the University of Utah for SERS analysis in conjunction with ELISA to detect an antigen associated with pancreatic cancer [2]. The SERS signal was generated from a reporter molecule complexed to both a gold nanoparticle and the target analyte in an otherwise classical lateral-flow or «sandwich» immunoassay (Figure 2). This is an incredibly accurate technique, enabling very sensitive detection – with potential for quantification – of the biomarker of interest.

Figure 2. Detection of an antigen associated with pancreatic cancer is possible with surface-enhanced Raman spectroscopy (SERS).

Rapid, Point-of-Care (POC) assay for femtogram-level detection of COVID-19

MIRA XTR, a handheld Raman spectrometer from Metrohm.
Figure 3. MIRA XTR, a handheld Raman spectrometer from Metrohm.

An alternate ELISA format was used by researchers at the University of Wyoming to detect antigen biomarkers associated with COVID-19 infection [4]. This study employed a magnetic nanoparticle-supported assay to concentrate the target biomarker in solution for subsequent SERS detection with MIRA XTR (Figure 3). It proved to be more sensitive than commercial lateral flow assays, was compatible with both solvent and saliva samples, could be adapted to new virus variants, and achieved highly sensitive POC diagnosis of COVID-19.

Lateral flow immunoassays provide relatively quick results, but they deliver nanogram-level detection and have quantification limitations. In comparison, the SERS-based ELISA is sensitive to femtogram quantities of antigen, with fast results at the POC with a commercial handheld Raman instrument.

Multiplex immunophenotyping of blood and breast cancer cells with Raman spectroscopy

Another study used MIRA DS to evaluate a portable SERS-based ELISA for immunophenotyping of different types of red blood and breast cancer cell surfaces [5]. Distinguishing healthy and diseased cells and detection of multiple biotargets in one sample can support informed treatment of different kinds of breast cancer.

This assay had «specificity, sensitivity, and repeatability for… immunophenotyping in different cell types [using] smaller analysis sample volume as compared to conventional... multiplex immunoassays,» and it was less «labor-intensive and technically straightforward to perform.» The authors praised MIRA’s Orbital Raster Scan for improving the sensitivity of their assay through a larger area of interrogation and spatially averaged measurements.

Traditional multiplexing flow assays can be limited by availability of different colored stains and interpretation of results. They are also associated with a large laboratory footprint. In contrast, this method based on handheld Raman spectroscopy offers the potential for accurate POC results with quick sample-to-result time, multiplexing capability, and a very compact instrument. 

Easy detection of enzymes with the electrochemical-SERS effect

The SPELECRAMAN638 instrument from Metrohm performs spectroelectrochemical Raman measurements using a 638 nm laser.
Figure 4. The SPELECRAMAN638 instrument from Metrohm performs spectroelectrochemical Raman measurements using a 638 nm laser.

A very different SERS technique for characterizing biological molecules has been reported by Metrohm [6]. Electrochemical SERS (EC-SERS) facilitates two experiments at once: electrochemical activation of SERS features of silver electrodes (Ag SPEs) followed by spectroscopic detection of the sample (with SPELECRAMAN638, Figure 4).

Here, the SERS substrate is generated in-situ from silver electrodes (screen-printed as well as conventional). This is accomplished in the presence of the analyte while under continuous interrogation with Raman to optimize detection of SERS-active species. The 638 nm excitation provides a SERS effect with good intensity, with less risk of sample damage and fluorescence.

Determining the structure of enzymes (and their role in disease) such as aldehyde dehydrogenase (ALDH) helps to understand diseases. With EC-SERS, application scientists defined previously unreported fingerprint Raman bands of ALDH in solution. Similarly, the redox states of cytochrome c provide information about electron transport across cell membranes [7]. Cytochrome c changes oxidation- and conformation-state during the EC experiment, and these redox states possess distinguishable SERS spectra.

Conclusion

Raman technology is truly being utilized in innovative ways. Research groups worldwide are applying its considerable benefits – including sensitivity, trace detection, small form-factor, and fast results – for detecting cancerous tissue, disease biomarkers, and disease-causing pathogens. The findings are incredibly exciting and promising!

[1] Lindtner, R. A.; Wurm, A.; Pirchner, E.; et al. Enhancing Bone Infection Diagnosis with Raman Handheld Spectroscopy: Pathogen Discrimination and Diagnostic Potential. IJMS 2023, 25 (1), 541. DOI:10.3390/ijms25010541

[2] Thomas, R.; Bakeev, K.; Claybourn, M.; Chimenti, R. The Use of Raman Spectroscopy in Cancer Diagnostics. Spectroscopy 2013, 28 (9), 36–43.

[3] Goonetilleke, K. S.; Siriwardena, A. K. Systematic Review of Carbohydrate Antigen (CA 19-9) as a Biochemical Marker in the Diagnosis of Pancreatic Cancer. Eur J Surg Oncol 2007, 33 (3), 266–270. DOI:10.1016/j.ejso.2006.10.004

[4] Antoine, D.; Mohammadi, M.; Vitt, M.; et al. Rapid, Point-of-Care ScFv-SERS Assay for Femtogram Level Detection of SARS-CoV-2. ACS Sens. 2022, 7 (3), 866–873. DOI:10.1021/acssensors.1c02664

[5] Wang, J.; Koo, K. M.; Trau, M. Tetraplex Immunophenotyping of Cell Surface Proteomes via Synthesized Plasmonic Nanotags and Portable Raman Spectroscopy. Anal. Chem. 2022, 94 (43), 14906–14916. DOI:10.1021/acs.analchem.2c02262

[6] Metrohm AG. Easy Detection of Enzymes with the Electrochemical-SERS Effect; AN-RA-008; Metrohm AG: Herisau, Switzerland, 2023.

[7] Brazhe, N. A.; Evlyukhin, A. B.; Goodilin, E. A.; et al. Probing Cytochrome c in Living Mitochondria with Surface-Enhanced Raman Spectroscopy. Sci Rep 2015, 5 (1), 13793. DOI:10.1038/srep13793

Your knowledge take-aways

Application Note: The Use of Raman Spectroscopy in the Field of Cancer Diagnostics

Application Note: Easy detection of enzymes with the electrochemical-SERS effect – Activation of enhanced features of silver electrodes helps obtain characteristic Raman bands of biological molecules

SERS trace detection solutions

Blog: Frequently Asked Questions (FAQ) about Raman spectroscopy: Theory and usage

Blog: Frequently Asked Questions (FAQ) about Raman spectroscopy: Applications

Blog: Handheld 785 nm Raman applications: Cutting edge material ID capabilities in your pocket

Blog: Raman spectroelectrochemistry from India to Spain: History and applications

Surface-Enhanced Raman Scattering (SERS) – Expanding the Limits of Conventional Raman Analysis

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Surface-Enhanced Raman Scattering, or SERS, is an anomalous enhancement of Raman scattering when molecules are adsorbed to gold or silver nanoparticles – this enhancement can be as large as 1e+7. The advantage of SERS for the analytical chemist lies in its ability to detect analyte concentrations of mg/L (ppm) and even µg/L (ppb), while classical Raman is limited to g/L (ppt). Metrohm Raman produces P-SERS assays in the form of nanoparticles printed onto substrates using inkjet technology. This method produces inexpensive test strips that exhibit exceptional stability and sensitivity. There are two markets that can be easily addressed with P-SERS: forensic analysis and food safety. This White Paper explains the mechanism of SERS and how it can be applied to handheld Raman analysis with Metrohm Raman MIRA systems.

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Gelwicks

Dr. Melissa Gelwicks

Marketing Specialist
Metrohm Raman (a division of Metrohm Spectro), Laramie, Wyoming (USA)

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