Electroactive 3D Porous Graphene Foam Impedimetric Biosensor for Ultrasensitive Detection of MCF‐7‐Cells‐Derived Extracellular Vesicles in Human Serum

ABSTRACT Efficient isolation and ultrasensitive detection of cancer cell‐derived extracellular vesicles (EVs), which carry molecular fingerprints of their parent cells, can enable precise early‐stage cancer diagnostics and prognostics. In this study, we developed a label‐free impedimetric biosensor using electroactive 3D porous graphene foam (3D‐PGF) electrodes to detect CD81 protein and MCF‐7 cells‐derived EVs in human serum. 1‐pyrenebutyric acid N‐hydroxysuccinimide ester (PYB‐NHS) was non‐covalently attached to the 3D‐PGF via π–π interactions without compromising structural integrity or electrocatalytic performance. Sensor selectivity was achieved by immobilizing anti‐CD81 antibodies onto the modified electrode, followed by quenching and blocking with ethanolamine and bovine serum albumin. MCF‐7 cells‐derived EVs were isolated by ultracentrifugation and characterized using nanoparticle tracking analysis and immunoaffinity plate‐based assay. Capture and detection of CD81‐ and MCF‐7‐derived EVs were evaluated using electrochemical impedance spectroscopy with a ferro/ferri redox mediator. The sensor demonstrated exceptional sensitivity, with limits of detection of 0.048 fg/mL for CD81 protein and ∼24 EVs/mL for MCF‐7 EVs. Performance was benchmarked against 2D graphene electrodes, highlighting structural and electrochemical advantages. The binder‐free 3D‐PGF combined with non‐covalent functionalization establishes a robust platform for selective, label‐free, and ultrasensitive detection of cancer‐cell‐derived EVs in human serum samples.