Avoiding Pre-Isolation Step in Exosome Analysis: Direct Isolation and Sensitive Detection of Exosomes Using Gold-Loaded Nanoporous Ferric Oxide Nanozymes

Most of the current exosome-analysis strategies are time-consuming and largely dependent on commercial extraction kit-based preisolation step, which requires extensive sample manipulations, costly isolation kits, reagents, tedious procedures, and sophisticated equipment and is prone to bias/artifacts. Herein we introduce a simple method for direct isolation and subsequent detection of a specific population of exosomes using an engineered superparamagnetic material with multifunctional properties, namely, gold-loaded ferric oxide nanocubes (Au-NPFe2O3NC). In this method, the Au-NPFe2O3NC were initially functionalized with a generic tetraspanin (exosomes-associated) antibody (i.e., CD63) and dispersed in sample fluids where they work as "dispersible nanocarriers" to capture the bulk population of exosomes. After magnetic collection and purification, Au-NPFe2O3NC-bound exosomes were transferred to the tissue-specific, antibody-modified, screen-printed electrode. As a proof of principle, we used a specific placental marker, placenta alkaline phosphatase (PLAP), to detect exosomes secreted from placental cells. The peroxidase-like activity of Au-NPFe2O3NC was then used to accomplish an enzyme-linked immunosorbent assay (ELISA)-based sensing protocol for naked-eye observation along with UV-visible and electrochemical detection of PLAP-specific exosomes present in placental cell-conditioned media. We demonstrated excellent agreement in analytical performance for the detection of placental cell-derived exosomes (i.e., linear dynamic range, 103-107 exosomes/mL; limit of detection, 103 exosomes/mL; relative standard deviation (%RSD) of <5.5% for n = 3) using with and without commercial "total exosome isolation kit"-based preisolation step. We envisage that this highly sensitive, rapid, and inexpensive assay could be useful in quantifying specific populations of exosomes for various clinical applications, focusing on pregnancy complications.