Ultrasensitive determination of exosomal CD63: A novel label-free and self-assembled electrochemical aptasensor for cancer diagnosis based on magnetic heterostructured Fe3O4/α-Fe2O3 nanosheets

Abstract

Cluster of Differentiation 63 (CD63) is one of the core marker proteins of exosomes and has been widely used as a biomarker for early screening of various cancers such as ovarian cancer and lung cancer, thus, its rapid and ultrasensitive detection is of great significance. This work presented a label-independent electrochemical aptamer (Apt)-based sensing platform for ultrasensitive exosomal CD63 determination. The core innovation lay in the development of magnetic Fe₃O₄/α-Fe₂O₃@Au-Apt complex probe, which streamlined operation via magnetic separation, improved antifouling performance through magnetic self-assembly technology, enhanced sensitivity by capitalizing on the superior conductivity of Au nanoparticles (AuNPs) and ensured specificity through the high-affinity interaction between aptamers and target. AuNPs were loaded on the surface of Fe₃O₄/α-Fe₂O₃ to improve its electrical conductivity while providing an anchor for the binding of thiol-modified Apt, and the bonding was achieved through Au-S bonds. After capturing the target, magnetic self-assembly was performed on the electrode surface for rapid detection. Under optimum conditions, the aptasensor demonstrated a linear response to CD63 between 100 pg/mL and 1 μg/mL, with lower detection limit reaching 13.4 pg/mL (S/N = 3). Furthermore, human serum samples validation achieved favorable recoveries (100.82–106.45 %), RSDs<3 %, confirming its robustness in complex matrices. Such an assay not only propels exosome-based liquid biopsy but also provides a novel strategy for diverse cancer related biomarkers detection through modular design of probe molecules and their synergistic incorporation with nanomaterials.