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 strategies for exosome analysis are time-consuming and largely dependent on pre-isolation step using a commercial extraction kit, which requires extensive sample manipulation, costly isolation, reagents, tedious procedures, and sophisticated equipment, and is prone to bias/artefacts. Herein we introduce a simple method for direct isolation and subsequent detection of a specific population of exosomes using an engineered superparamagnetic material with multi-functional 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 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 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 the pre-isolation step with commercial "total exosome isolation kit". 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.