Surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS) in label-free characterization of erythrocyte membranes and extracellular vesicles at the nano-scale and molecular level.

The manuscript presents the potential of surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS) for label-free characterization of extracellular microvesicles (EVs) and their isolated membranes derived from red blood cells (RBCs) at the nanoscale and at the single-molecule level, providing detection of a few individual amino acids, protein and lipid membrane compartments. The study shows future directions for research, such as investigating the use of the mentioned techniques for the detection and diagnosis of diseases. We demonstrate that SERS and TERS are powerful techniques for identifying the biochemical composition of EVs and their membranes, allowing the detection of small molecules, lipids, and proteins. Furthermore, extracellular vesicles released from red blood cells (REVs) can be broadly classified into exosomes, microvesicles, and apoptotic bodies, based on their size and biogenesis pathways. Our study specifically focuses on microvesicles that range from 100 to 1000 nanometres in diameter, as presented in AFM images. Using SERS and TERS spectra obtained for REVs and their membranes, we were able to characterize the chemical and structural properties of microvesicle membranes with high sensitivity and specificity. This information may help better distinguish and categorize different types of EVs, leading to a better understanding of their functions and potential biomedical applications.

Sulfhemoglobin under the spotlight - Detection and characterization of SHb and HbFeIII-SH.

Sulfhemoglobinemia is an incurable disease caused by an overdose of sulfur-containing drugs with oxidizing properties. Its diagnosis remains hindered due to the similarity of symptoms to other pathological state - methemoglobinemia, as well as contradictory information on the structure and characteristics of sulfhemoglobin. Herein, we present sulfhemoglobinemia model on living functional human erythrocytes, designed to recreate processes which could take place in a patient body in order to complement missing information and highlight distinctiveness of two hemoglobin (Hb) adducts formed after interaction with sulfur donors. Employed techniques, UV-Vis absorption, Raman, Fourier transformed infrared (FT-IR) and electronic circular dichroism (ECD) spectroscopies, allowed to distinguish and characterize Hb adduct with sulfur atom bounded directly to the iron ion (HbFeIII-SH), and irreversibly connected to the porphyrin ring (SHb - sulfhemoglobin). Presented herein results provided also new evidence on formation of both these hemoglobin adducts inside functional erythrocytes under oxidative conditions and during sulfur-containing drug presence, what can be further translated into future physiological studies. Moreover, we found that sulfur attachment to the porphyrin ring altered Hb structure and lead to changes in protein packing inside RBCs, eventually. Interestingly, measurement of blood drop smear by Raman spectroscopy occurred the most accurate method to differentiate HbFeIII-SH and SHb, indicating potential of this technique in sulfhemoglobinemia diagnosis.

Spectroscopic Signature of Red Blood Cells in a D-Galactose-Induced Accelerated Aging Model.

This work presents a semi-quantitative spectroscopic approach, including FTIR-ATR and Raman spectroscopies, for the biochemical analysis of red blood cells (RBCs) supported by the biochemical, morphological and rheological reference techniques. This multi-modal approach provided the description of the RBC alterations at the molecular level in a model of accelerated aging induced by administration of D-galactose (D-gal), in comparison to natural aging. Such an approach allowed to conclude that most age-related biochemical RBC membrane changes (a decrease in lipid unsaturation and the level of phospholipids, or an increase in acyl chain shortening) as well as alterations in the morphological parameters and RBC deformability are well reflected in the D-gal model of accelerated aging. Similarly, as in natural aging, a decrease in LDL level in blood plasma and no changes in the fraction of glucose, creatinine, total cholesterol, HDL, iron, or triglycerides were observed during the course of accelerated aging. Contrary to natural aging, the D-gal model led to an increase in cholesterol esters and the fraction of total esterified lipids in RBC membranes, and evoked significant changes in the secondary structure of the membrane proteins. Moreover, a significant decrease in the phosphorous level of blood plasma was specific for the D-gal model. On the other hand, natural aging induced stronger changes in the secondary structures of the proteins of the RBCs' interior. This work proves that research on the aging mechanism, especially in circulation-related diseases, should employ the D-gal model with caution. Nonetheless, the D-gal model enables to imitate age-related rheological alterations in RBCs, although they are partially derived from different changes observed in the RBC membrane at the molecular level.