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<p>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&#xa0;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 × 10<sup>3</sup> to 4.0 × 10<sup>7</sup> particles/mL (colorimetric) and 4.0 × 10<sup>2</sup> to 4.0 × 10<sup>6</sup> particles/mL (fluorescence), and low detection limits of 1.78 × 10<sup>3</sup> 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.</p> Graphical Abstract <p>A two-signal aptasensor was elegantly designed for sensitive exosome detection. Magnetic&#xa0;beads (MBs) modified with CD63 antibodies were employed for efficient exosome capture&#xa0;and purification. The CD63 aptamers and G4-quadruplexes (G4) in the rolling circle&#xa0;amplification (RCA) products formed a sophisticated sandwich structure with exosomes. By&#xa0;integrating with Hemin and NMM, the system generated proportional colorimetric and&#xa0;fluorescence signals, offering a reliable and precise method for exosome quantification.</p>

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

  • 期刊论文

  • 发表日期：

    2025年10月3日

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  • Xu Sun,
  • Qing Dong,
  • Xuemei Zhang,
  • Fan Feng,
  • Junliang Chang,
  • Dan Li,
  • Jin Wang,
  • Erkang Wang

摘要

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.

Graphical Abstract

A two-signal aptasensor was elegantly designed for sensitive exosome detection. Magnetic beads (MBs) modified with CD63 antibodies were employed for efficient exosome capture and purification. The CD63 aptamers and G4-quadruplexes (G4) in the rolling circle amplification (RCA) products formed a sophisticated sandwich structure with exosomes. By integrating with Hemin and NMM, the system generated proportional colorimetric and fluorescence signals, offering a reliable and precise method for exosome quantification.