Abstract
Exosomes are cell-derived nanovesicles released into biological fluids, which are involved in cell-to-cell communication. The analysis of the content and the surface of the exosomes allow conclusions about the cells they are originating from and the underlying condition, pathology or disease. Therefore, the exosomes are currently considered good candidates as biomarkers to improve the current methods for clinical diagnosis, including cancer. However, due to their low concentration, conventional procedures for exosome detection including biosensing usually require relatively large sample volumes and involve preliminary purification and preconcentration steps by ultracentrifugation. In this paper, the immunomagnetic separation is presented as an alternative method for the specific isolation of exosomes in serum. To achieve that, a rational study of the surface proteins in exosomes, which can be recognized by magnetic particles, is presented. The characterization was performed in exosomes obtained from cell culture supernatants of MCF7, MDA-MB-231 and SKBR3 breast cancer cell lines, including TEM and nanoparticle tracking analysis (NTA). For the specific characterization by flow cytometry and confocal microscopy, different commercial antibodies against selected receptors were used, including the general tetraspanins CD9, CD63 and CD81, and cancer-related receptors (CD24, CD44, CD54, CD326 and CD340). The effect of the serum matrix on the immunomagnetic separation was then carefully evaluated by spiking the exosomes in depleted human serum. Based on this study, the exosomes were preconcentrated by immunomagnetic separation on antiCD81-modified magnetic particles in order to achieve further magnetic actuation on the surface of the electrode for the electrochemical readout. The performance of this approach is discussed and compared with classical characterization methods.
Highlights
Breast cancer is a highly lethal malignancy and the most commonly diagnosed cancer among women (WHO), with an estimated over 2 million new cases in 2018 [1]
The most important feature that should be considered to simplify the analytical procedure for the detection of exosomes at low concentration levels involves novel solid-phase separation methods to avoid ultracentrifugation
We demonstrated that particle-based magnetic enrichment simplifies exosomes isolation and can be coupled with emerging technologies as is the case with electrochemical immunosensing
Summary
Breast cancer is a highly lethal malignancy and the most commonly diagnosed cancer among women (WHO), with an estimated over 2 million new cases in 2018 [1]. Exosomes are extracellular nanovesicles of 30–150 nm released by all types of cells during fusion of the multivesicular endosomes (MVEs) with the plasmatic membrane. One of the most remarkable features is that they are present in all the biological fluids, such as blood [5], saliva [6] and urine [7], among others Their easy accessibility is one of the most compelling reasons for developing exosomes as clinical biomarkers. Another striking characteristic is their molecular cargo, which can be useful for diagnosis and prognosis of several diseases and conditions. They carry specific surface markers that indicate their cell signatures, including surface proteins such as proteins related to transport and fusion (e.g., flotillin, caveolin-1), tetraspanins (e.g., CD9, CD63, CD81), heat shock proteins (e.g., Hsp90) and lipid-related proteins, as well as micro RNA (miRNA), messenger
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