Neurodegenerative diseases suppose an important public health issue in our aging society, but the aetiology of these disorders is quite complex and therapies are sometimes limited. Thus, there is a need to discover reliable biomarkers, especially in the early stages of diseases. In this vein, one of the most promising research fields of the last decade in biomedicine is the study of the secretory activity of cells [1]. Cells secrete extracellular vesicles (EVs) that serve as a mechanism of intercellular communication, key in cell signalling and onset of several diseases, and hence EVs can be considered as a valuable source of target biomarkers. It should be stated that EVs are secreted to the bloodstream, so analysing EVs purified from blood serum is a promising option, as it is considered one of the biggest sources of biomarkers in the organism, and serum extraction is minimally invasive. However, biological fluids are not available in all cases, so a suitable alternative is to work with in vitro cellular models.

Conventional nebulization ICP-MS stands out as a highly versatile tool for trace element research, owing to its remarkable attributes, which encompass exceptional limits of detection, minimal matrix effects, and a broad linear dynamic range. Despite the numerous potential advantages offered by ICP-MS, there is a lack of methodologies for the analysis of metals and target molecules in EVs [2]. This lack of methodologies can be attributed to the challenges associated with the limited sample volume available and the extremely low levels of analytes in EVs.

In this work, we present the determination of endogenous metals and target proteins in EVs isolated from two type of biological samples (cell culture media and serum) by ICP-MS. On one hand, endogenous Fe, Cu, and Zn in exosomes (<200 nm EVs) secreted by an in vitro model of the human retinal pigment epithelium (HRPEsv cell line; as a model of ocular neurodegenerative diseases) was studied, both in control and pro-inflammatory stress conditions. Two protocols for exosomes isolation from RPE cell culture media, based on differential centrifugation and precipitation were evaluated. Additionally, considering that the analysis of low volume samples is still challenging by ICP-MS, three sample introduction set-ups were investigated for the analysis of exosomes by ICP-MS, including a micronebulizer and two interfaces designed for single cell analysis (as total consumption setups). On the other hand, we have also focused on the determination of specific target proteins in EVs from mouse serum samples. For the analysis of biomolecules by ICP-MS, a direct competitive immunoassay protocol was developed using two different labels (Au nanoparticles; AuNPs and Au nanoclusters; AuNCs) in order to select the most appropriate in terms of reproducibility and sensitivity. As a model case, CD81 (membrane bound protein of the tetraspanin superfamily) has been selected as the target protein in EVs purified from mouse serum samples.

References

[1] R. Kalluri R, V. S. LeBleu Science 2020; 367(6478), eaau6977.

[2] J. Martínez-García, A. Villa-Vázquez, B. Fernández, H. González-Iglesias, R. Pereiro Anal. Bioanal. Chem. 2024; 416, 2595–2604.

Acknowledgements: This work was financially supported through the coordinated project PID2022-137319OB (-C21 and -C22) funded by MCIN/AEI/10.13039/501100011033. J. Martínez-García acknowledges the Severo Ochoa Grant with ref. BP21-041 (Principality of Asturias, Spain).