On-Capillary Surface-Enhanced Raman Spectroscopy for Determination of Glutathione in Whole Blood

November 1, 2018

On-capillary surface-enhanced Raman spectroscopy (SERS) is showing dramatic potential for analysis of human whole blood constituents using microsampling. A group of researchers has recently published a method to measure glutathione (GSH) in a 2 μL sample of human whole blood. This exciting development could lead to rapid point-of-care analysis of other essential blood components. We recently interviewed Julia Kuligowski of the Health Research Institute La Fe, in Valencia, Spain, and Guillermo Quintas, of the LEITAT Technological Center in Barcelona, about this research.

On-capillary surface-enhanced Raman spectroscopy (SERS) is showing dramatic potential for analysis of human whole blood constituents using microsampling. A group of researchers has recently published a method to measure glutathione (GSH) in a 2 μL sample of human whole blood. This exciting development could lead to rapid point-of-care analysis of other essential blood components. We recently interviewed Julia Kuligowski of the Health Research Institute La Fe, in Valencia, Spain, and Guillermo Quintas, of the LEITAT Technological Center in Barcelona, about this research.

You have developed a new on-capillary method for measuring glutathione in whole blood using surface-enhanced Raman spectroscopy (SERS). What prompted you to investigate this problem? What is unique about your approach?

Glutathione is the most abundant antioxidant in cells and tissues and it has been used for the in-vivo assessment of oxidative stress for decades. However, it is very difficult to obtain accurate concentrations employing conventional analytical approaches due to stability issues of glutathione. The fast and direct approach for the determination of GSH that we have developed employing SERS allows us to circumvent problems related to sample processing and storage (1). 

 

Why is the determination of glutathione important, and why are these analysis results needed for point-of-care use?

Glutathione is a well-known biomarker for oxidative stress, which is involved in many pathologies such as cancer, neurodegenerative diseases, diabetes, and cardiovascular disease as well as physiological processes such as aging. The measurement of glutathione in blood and tissues allows us to quickly assess the global redox balance. Hence, the determination of glutathione is potentially of interest in many different clinical settings. Yet a reliable, cheap, and fast method that allows widespread use of this biomarker has been lacking and consequently the use of glutathione has been primarily limited to research studies. For example, in neonatal care, oxygen therapy is frequently employed, and it could potentially be interesting to measure oxidative damage during the stay of a newborn in the neonatal intensive care unit (NICU), but this requires access to a device that can generate results fast and in a minimally invasive manner.

In short, we believe that the reason for the limited use of glutathione determinations in the clinical setting is the lack of a straightforward analytical solution. Now, employing SERS, we are trying to fill this gap and eventually this technique will allow the development of a point-of-care device.

 

What are the advantages of using SERS over other methods for glutathioneanalysis in whole blood? And specifically, why is SERS preferable to using standard clinical methods, which have been proven reliable over time?

By providing fast and direct results, SERS allows us to obtain more accurate results by avoiding glutathione degradation. In the past, methods such as enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS)-based assays have been employed, but they all have suffered from important drawbacks for their application in the clinical environment. To provide reliable results, these methods required laborious sample preparation, expensive equipment, and an experienced scientist. For example, a well-established approach for sample conservation is the use of a chemical agent, N-ethylmaleimide, that acts by blocking thiol groups and therefore avoids the oxidation of glutathione. The downside is that the reagent has to be added to blood samples immediately, which very often is not feasible in clinical studies.

 

What are the unique aspects of sample preparation for your method of implementing SERS, and why did you choose that approach for a 2-μL sample of whole blood? What enhancement in signal did you achieve using SERS over standard Raman spectra? Did this level of signal enhancement surprise you?

The advantage of the sample preparation procedure employed in our method is that the use of perchloric acid helps us eliminate proteins that would inhibit generation of the SERS signals while at the same time breaking open the erythrocytes, which is important because most of glutathione molecules are located inside the red blood cells and have to be released first for analysis.

Another important aspect is the use of an isotopically labeled internal standard. The main reason why there is very little reporting in the scientific literature on the use of SERS for quantitative measurements is that it is very difficult to obtain reproducible results. Here, we use the relative signal of glutathione and the internal standard to compensate for fluctuations of the absolute detected signal intensities.

The reason why we chose to use 2 μL is that we wanted to detect glutathione in very small amounts of whole blood in view of the application of our method in the NICU. If you intend to carry out serial determinations in newborns, it is very important that the amount of withdrawn blood be as small as possible so that the test will not affect the patient. At the same time, the use of small volumes is, we think, an advantage for the implementation of our method in a fully automatized system because it is potentially compatible with lab-on-a-chip approaches.

The signal enhancement that we achieved by using SERS as compared to conventional Raman spectroscopy was amazing: With SERS we were actually able to see glutathione at physiologically relevant concentrations whereas with classical Raman spectroscopy no distinguishable peaks were detected. However, what surprised us even more is the specificity of the obtained signal. We tried a whole range of biomolecules with similar structural elements and none of them were SERS-active in the tested conditions.

 

What do you anticipate will be your next area of research in this field, and what will be your next steps in advancing this work? Do you anticipate any regulatory concerns to implement the methods you are developing in a clinical or hospital setting?

Our next project is focusing on the simultaneous determination of oxidized glutathione (GSSG). The GSH/GSSG ratio is widely employed and certainly of interest for our field of application. Also, we want to automate the whole procedure and miniaturize the detection system, moving toward a handheld, portable device. So far there have been no noteworthy concerns for obtaining the necessary permissions for testing of our method, however we are still at a very early stage. Legal and regulatory aspects will certainly become a relevant issue once we decide to move forward from a research project aiming at the development of a new technology to the implementation of a SERS-based tool in the clinical practice.

 

Reference

(1) Á. Sánchez-Illana, F. Mayr, D. Cuesta-García, J.D. Piñeiro-Ramos, A. Cantarero, M. de la Guardia, M. Vento, B. Lendl, G. Quintás, and J. Kuligowski, Anal. Chem. 90(15), 9093–9100 (2018). doi: 10.1021/acs.analchem.8b01492.

Julia Kuligowski, PhD, is a postdoctoral researcher at the Health Research Institute La Fe, in Valencia, Spain, where her main research focus is on clinical metabolomics and advanced data analysis tools. She studied biotechnological processes at the University of Applied Sciences Wiener Neustadt, Tulln (Austria) and was trained as an analytical chemist at the Vienna University of Technology (Austria) and the University of Valencia (Spain) where she earned her PhD in 2011. As part of the Neonatal Research Unit, her research focused on the quantification of metabolites and biomarkers in biological samples obtained from clinical trials and animal models employing mass spectrometry–based techniques and vibrational spectroscopy. At present she continues her research activities in the field of neonatology focusing on the discovery of molecular biomarkers for an early, minimally invasive assessment of brain injury secondary to hypoxic-ischemic encephalopathy and the impact of nutrition on growth and development of preterm infants.

 

Guillermo Quintas, PhD, is a senior researcher at the LEITAT Technological Center in Barcelona, Spain. After completing his PhD at the University of Valencia (Spain) in 2004 he obtained a post-doctoral fellowship at the Public Health Laboratory Valencia (Spain), the Vienna University of Technology (Austria), and the University of Valencia, where his research focused on hyphenated systems and infrared spectroscopy. In 2009 he made the transition to a position as senior researcher in Advancell (Barcelona, Spain) and at the Analytical Unit of the Health Research Institute La Fe (Valencia) where his activities were focused on providing tailored analytical solutions for clinical research studies aiming at biomarker discovery and validation. In 2011, he moved to the Bio in vitro division of LEITAT. Currently his main research is directed to the joint analysis of information from multiple platforms (miRNA, metabolomics, and spectroscopy) and clinical data developing novel chemometric approaches.