An ultrasensitive electrochemical aptasensor for the determination of tumor exosomes based on click chemistry

Exosomes, lipid bilayer membrane vesicles, can guide various pathological and physiological processes. However, reliable, convenient and sensitive methods for exosome determination for early cancer diagnosis are still technically challenging. Herein, an electrochemical aptasensor based on click chemistry and the DNA hybridization chain reaction (HCR) for signal amplification has been developed for the ultrasensitive detection of tumor exosomes. CD63 aptamer was first immobilized on a glassy carbon electrode for capturing exosomes, and 4-oxo-2-nonenal alkyne (alkynyl-4-ONE) molecules, functionalized lipid electrophiles, were conjugated to the exosomes via the reaction of amino and aldehyde groups. Azide-labeled DNA probe as an anchor was then connected to the exosomes by copper (I)-catalyzed click chemistry. Signal amplification was achieved by HCR, and the numerous linked horseradish peroxidase (HRP) molecules could catalyze the reaction of o-phenylenediamine (OPD) and H₂O₂. The concentration of exosomes could be quantified by monitoring the electrochemical reduction current of 2,3-diaminophenazine (DAP). Under the optimal conditions, this method allowed the sensitive detection of exosomes in the range of 1.12 × 10² to 1.12 × 10⁸ particles/μL with a limit of detection (LOD) of 96 particles/μL. Furthermore, the present assay enabled sensitive and accurate quantification of exosomes in human serum, and it has high potential for exosome analysis in clinical samples.