Quantification of Serum Exosome Biomarkers Using 3D Nanoporous Gold and Spectrophotometry.

Abstract

Tumor-derived exosomes may provide biomarkers for cancer treatment. Using sputtering technology, an affinity-based device to capture exosomes was developed using nanoporous substrate (NPG)-coated silicon microscopy. Immunology-based techniques detect and purify exosomes using gold coating with a specific antigen. Inverted fluorescent microscopy was used to detect target exosomes quantitatively utilizing fluorescent nanospheres as the label. We quantified the expression of CD63 surface protein markers on exosomes from conditioned culture media of breast cancer cells. The exosomes that targeted specific proteins with controls were statistically analyzed and compared to those that targeted non-specific proteins. Results from SEM showed that the exosomes were circular, between 30 and 150 nanometers in size. The porous gold substrates captured more exosomes than the nonporous substrates. Nitric acid treatments at different times resulted in a variety of pore sizes. Despite the increase in the size of the pores, the number of exosomes found in the porous gold substrate treated for 10 min nearly doubled compared to the one treated for 5 min. In this work, a fluorescence biosensor was developed to detect breast cancer exosomes using nanoporous gold substrates (NPG). Assay and model exosomes of specific breast cancer cells showed that exosomes exhibit diagnostic surface protein markers, reflecting the protein profile of their parent cells. Furthermore, the specific binding between the exosome surface antibodies and the targets identified the CD63 biomarkers on the exosome, suggesting these markers’ diagnostic potential. This study can accelerate exosome research in determining tumor-related exosomes and develop novel cancer diagnostic methods.