Bottom-Up Signal Boosting with Fractal Nanostructuring and Primer Exchange Reaction for Ultrasensitive Detection of Cancerous Exosomes

Exosomes are emerging as promising biomarkers for cancer diagnosis, yet sensitive and accurate quantification of tumor-derived exosomes remains a challenge. Here, we report an ultrasensitive and specific exosome sensor (NPExo) that initially leverages hierarchical nanostructuring array and primer exchange reaction (PER) for quantitation of cancerous exosomes. This NPExo uses a high-curvature nanostructuring array (bottom) fabricated by single-step electrodeposition to enhance capturing of the target exosomes. The immuno-captured exosome thus provides abundant membrane sites to insert numerous cholesterol-DNA probes with a density much higher than that by immune pairing, which further allows PER-based DNA extension to assemble enzyme concatemers (up) for signal amplification. Such a bottom-up signal-boosting design imparts NPExo with ultrahigh sensitivity up to 75 particles/mL (i.e., <1 exosome per 10 μL) and a broad dynamic range spanning 6 orders of magnitude. Furthermore, our sensor allows monitoring subtle exosomal phenotypic transition and shows high accuracy in discrimination of liver cancer patients from healthy donors via blood samples, suggesting the great potential of NPExo as a promising tool in clinical diagnostics.