Automatic Electrochemiluminescence Method for the Detection of Cancerous Exosomes Incorporating Specific Aptamer-Magnetic Beads and Signal Nanoprobes

Exosomes, as an emerging biomarker, have exhibited remarkable promise in early cancer diagnosis. Here, a highly sensitive, selective, and automatic electrochemiluminescence (ECL) method for the detection of cancerous exosomes was developed. Specific aptamer-(EK)₄ peptide-tagged magnetic beads (MBs-(EK)₄-aptamer) were designed as a magnetic capture probe in which the (EK)₄ peptide was used to reduce the steric binding hindrance of cancerous exosomes with a specific aptamer. One new universal ECL signal nanoprobe (CD9 Ab-PEG@SiO₂ϵRu(bpy)₃²⁺) was designed and synthesized by using microporous SiO₂ nanoparticles as the carrier for loading ECL reagent Ru(bpy)₃²⁺, polyethylene glycol (PEG) layer, and anticluster of differentiation 9 antibody (CD9 Ab). A "sandwich" biocomplex was formed on the surface of the magnetic capture probe after mixing the capture probe, target exosomes, and ECL signal nanoprobe, and then it was introduced into an automated ECL analyzer for rapid and automatic ECL measurement. It was found that the designed signal nanoprobe shows a 270-fold improvement in the signal-to-noise ratio than that of the ruthenium complex-labeled CD9 antibody signal probe. The relative ECL intensity was proportional to MCF-7 exosomes as a model in the range of 10² to 10⁴ particle/μL, with a detection limit of 11 particle/μL. Furthermore, the ECL method was employed to discriminate cancerous exosomes based on fingerprint responses using the designed multiple magnetic capture probes and the universal ECL signal nanoprobe. This work demonstrates that the utilization of a designed automated ECL tactic using the MBs-(EK)₄-aptamer capture probe and the CD9 Ab-PEG@SiO₂ϵRu(bpy)₃²⁺ signal nanoprobe will provide a unique and robust method for the detection and discrimination of cancerous exosomes.