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.

Interfacial DNA self-assembly is fundamental to solid nucleic acid biosensors, whereas how to improve the signal-to-noise ratio has always been a challenge, especially in the charge-based electrochemical DNA sensors because of the large noise from the negatively charged DNA capture probes. Here, we report a DNA framework-reversed signal-gain strategy through background-to-signal transition for ultrasensitive and highly specific electrical detection of microRNAs (miRNAs) in blood. By using a model of enzyme-catalyzed deposition of conductive molecules (polyaniline) targeting to DNA, we observed the highest signal contribution per unit area by the highly charged three-dimensional (3D) tetrahedral DNA framework probe, relative to the modest of two-dimensional (2D) polyA probe and the lowest of one-dimensional (1D) single-stranded (ss)­DNA probe, suggesting the positive correlation of background DNA charge with signal enhancement. Using such an effective signal-transition design, the DNA framework-based electrochemical sensor achieves ultrasensitive miRNAs detection with sensitivity up to 0.29 fM (at least 10-fold higher than that with 1D ssDNA or 2D polyA probes) and high specificity with single-base resolution. More importantly, this high-performance sensor allows for a generalized sandwich detection of tumor-associated miRNAs in the complex matrices (multiple cell lysates and blood serum) and further distinguishes the tumor patients (e.g., breast, lung, and liver cancer) from the normal individuals. These advantages signify the promise of this miRNA sensor as a versatile tool in precision diagnosis.

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