Enabling Sensitive Quantification of Exosomes Combining Aptamer-Based Rolling Circle Amplification and Silver Nanoparticles

Exosomes carry various biological information and are abundant in body fluids, making them a promising noninvasive biomarker for disease diagnosis and prognosis. However, current detection methods have limitations in sensitivity, specificity, and cost effectiveness, hindering their clinical application. To address these challenges, we have developed a fast, accurate, and cost-effective method for detecting exosomes with high sensitivity and specificity, making it ideal for clinical applications. Clusters of differentiation 63 (CD63) aptamer with its complementary DNA (CD63 aptamer/cDNA) linked to streptavidin-coated magnetic beads (SA-MBs) are used as a capture probe. Exosomes with CD63 proteins can bind to the aptamer and release the cDNA, which initiates rolling circle amplification (RCA) to magnify the cDNA copies. The negatively charged RCA products induce the aggregation of positively charged spermine-modified silver nanoparticles (AgNPs) through electrostatic attraction. The aggregation of AgNPs can be observed visually with the naked eye or quantitatively analyzed using ultraviolet-visible (UV-vis) spectroscopy to determine the concentration of exosomes, with limits of detection of 4.0 × 10⁴ particles/mL for visual observation and 800 particles/mL for UV-vis spectroscopy, respectively. The method has also been demonstrated for detecting the exosomes in serum samples, indicating its potential for clinical use in liquid biopsy.