A two-signal aptasensor for sensitive exosome detection via rolling circle amplification and G-quadruplex structure.

Exosomes play a pivotal role in cancer diagnosis and therapy, yet their clinical application faces significant technical challenges. To address this, we developed a sensitive, aptamer-based platform that integrates nucleic acid amplification and a G-quadruplex (G4) with two-signal output for exosome detection. Our approach employs CD63 antibody-conjugated magnetic beads (MBs) for efficient exosome capture and purification. The captured exosomes are then bound by CD63-specific aptamers embedded in rolling circle amplification (RCA) products, forming a sandwich complex with the MBs. The RCA products also contain G4 repeats, enabling two-signal output through interaction with Fe(III)-protoporphyrin IX (Hemin) and N-methylporphyrin-dipropionic acid IX (NMM). The G4-Hemin complex catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), producing a colorimetric signal, while the G4-NMM enhances fluorescence emission. This detection system achieves high sensitivity, with detection ranges of 4.0 × 10³ to 4.0 × 10⁷ particles/mL (colorimetric) and 4.0 × 10² to 4.0 × 10⁶ particles/mL (fluorescence), and low detection limits of 1.78 × 10³ particles/mL (colorimetric) and 96 particles/mL (fluorescence). Demonstrating high performance in complex media (10% exosome-depleted fetal bovine serum, FBS), this biosensor achieves sensitivity comparable to current methods at a low cost ($5.19 per test). This combination of robustness, sensitivity, and affordability makes it a highly promising platform for clinical exosome-based diagnostics.