Dual rolling circle amplification-enabled ultrasensitive multiplex detection of exosome biomarkers using electrochemical aptasensors.

Breast cancer is one of the leading causes of cancer-related death. An effective diagnostic system that enables early cancer detection is required for timely diagnosis and better treatment outcomes. Here, we developed an ultrasensitive electrochemical aptasensor for the multiplex detection of exosome biomarkers based on the electrochemical signals of metal ions. Specifically, a screen-printed carbon electrode (SPCE) was first modified with a multi-walled carbon nanotube (MWCNT), ionic liquid (IL), and chitosan (CHT) composite, and then gold nanoparticles (GNPs) were deposited via electrodeposition (GNPs/MWCNT-IL-CHT). To capture target exosomes, an aptamer specific for CD63, the universal exosome surface protein, was immobilized on the GNPs/MWCNT-IL-CHT/SPCE. When EpCAM or HER-2 positive exosomes were present in the sample, they could bind to EpCAM or HER-2 aptamers with primer sequences that acted as a rolling circle amplification reaction initiator, thereby generating numerous poly-guanine and poly-thymine repeats of a metal ion binding sequence, which produced strong electrochemical signals upon complexation with copper and lead ions. Using the proposed, multiplex exosome analysis system, EpCAM- and HER-2-positive exosomes were simultaneously detected with high specificity and a detection limit of 1 particle mL. In addition, its clinical applicability was validated via spike-and-recovery experiments using human serum samples.