Exosomes (EVs) preserve microRNAs (miRNAs) that report the condition of their parent cells, making miRNA a readout of tissue state. Enriching EVs from a defined source tissue tightens the match between the miRNA profile and that tissue, enhancing biological fidelity. However, heterogeneous collection and downstream EV processing often blur closely related miRNAs, obscuring disease-causing species and undermining early detection and diagnostic confidence. Moreover, given the low copy number of miRNAs per EV, bulk workflows demand large biofluid volumes and incur stepwise sample loss, without signal amplification, assays yield reduced sensitivity and reproducibility. This motivates target-selective capture and single-exosome sensing with amplification to improve throughput and specificity of miRNA-based detection. We propose a transmembrane sensor nanoarray that enables rapid, sensitive, and specific quantification of EV miRNA at single-vesicle resolution. The platform embeds cholesterol-labeled DNA hairpins across vesicle membranes to hybridize target miRNA, binding is reported by fluorescence, while an isothermal amplification cascade boosts signal from low-abundance targets. To benchmark insertion, vesicles (∼100 nm) were loaded with Cy3-labeled DNA, then Cy5-labeled, cholesterol-tagged hairpin sensors were added. Total internal reflection fluorescence (TIRF) imaging showed a 4.5× increase in Cy3-Cy5 colocalization with sensors, indicating hairpin insertion. Confocal fluorescence correlation measurements detected hairpin-vesicle complex formation, consistent with a larger hydrodynamic radius and not nonspecific adsorption. Next, biomarker detection will be evaluated with synthetic vesicles, using hybridization chain reaction and TIRF imaging to assess sensitivity. Then, pancreatic cancer derived exosomes will be immobilized on a DNA-origami nanoarray for high-throughput, specific, single-exosome quantification. Operating on intact vesicles, we hypothesize that cholesterol-tagged DNA hairpins will support single-vesicle readouts to resolve exosomal miRNA heterogeneity. If successful, this cost-effective system will deliver a high-specificity, ultrasensitive, and rapid liquid-biopsy approach for early disease detection without invasive tissue biopsies.
Chopade et al. (Sun,) studied this question.