Quantification of Exosome Based on a Copper-Mediated Signal Amplification Strategy

Exosomes, a class of small extracellular vesicles, play important roles in various physiological and pathological processes by serving as vehicles for transferring and delivering membrane and cytosolic molecules between cells. Since exosomes widely exist in various body fluids and carry molecular information on their originating cells, they are being regarded as potential noninvasive biomarkers. Nevertheless, the development of convenient and quantitative exosome analysis methods is still technically challenging. Here, we present a low-cost assay for direct capture and rapid detection of exosomes based on a copper-mediated signal amplification strategy. The assay involves three steps. First, bulk nanovesicles are magnetically captured by cholesterol-modified magnetic beads (MB) via hydrophobic interaction between cholesterol moieties and lipid membranes. Second, bead-binding nanovesicles of exosomes with a specific membrane protein are anchored with aptamer-modified copper oxide nanoparticles (CuO NPs) to form sandwich complexes (MB-exosome-CuO NP). Third, the resultant sandwich complexes are dissolved by acidolysis to turn CuO NP into copper(II) ions (Cu²⁺), which can be reduced to fluorescent copper nanoparticles (CuNPs) by sodium ascorbate in the presence of poly(thymine). The fluorescence emission of CuNPs increases with the increase of Cu²⁺ concentration, which is directly proportional to the concentration of exosomes. Our method allows quantitative analysis of exosomes in the range of 7.5 × 10⁴ to 1.5 × 10⁷ particles/μL with a detection of limit of 4.8 × 10⁴ particles/μL in biological sample. The total working time is about 2 h. The assay has the potential to be a simple and cost-effective method for routine exosome analysis in biological samples.