DNA Four-Way Junction-Driven Dual Rolling Circle Amplification Aptasensor for Ultra-Sensitive Detection of Tumor-Derived Exosomes

Abstract

There is an urgent need to accurately quantify tumor-derived exosomes, which have emerged as promising non-invasive tumor diagnostic biomarkers. Herein, a bispecific-aptamer sandwich-type gold nanoparticles-modified electrochemical aptasensor was developed based on a four-way-junction (4-WJ) triggered dual rolling circle amplification (RCA)-assisted methylene blue/guanine (MB/G) quadruplex strategy for extremely specific and sensitive tumor-derived exosome detection. This aptamer/exosome/aptamer sandwich-type aptasensor contains a CD63-specific aptamer and a cancerous mucin-1 (MUC1) protein-specific aptamer. When confronted with target exosomes, the CD63 aptamer modified on a gold electrode captured the targets. After that, the sandwich-type aptasensor was formed with the addition of the MUC1 aptamer. The sequence at the 3′-end of the MUC1 aptamer enabled the formation of a DNA 4-WJ with the help of a molecular beacon probe and a binary DNA probe. Subsequently, a dual-RCA reaction was triggered by binding to two cytosine-rich circle DNA templates at both ends of the 4-WJ structure. Ultimately, dual-RCA products containing multiple G-quadruplex conformations were generated with the assistance of K+ to trap abundant MB indicators to amplify the electrochemical signal. Our proposed aptasensor exhibited high specificity and sensitivity toward MCF-7-derived exosomes, with a detection limit of 20 particles/mL and a dynamic linear range of 1 × 102 to 1 × 107 particles/mL. Moreover, our aptasensor showed excellent repeatability, stability, and applicability in clinical settings. In conclusion, our electrochemical aptasensor is expected to become a universal platform for detecting other specific aptamer-based targets in biomedical and bioanalytical applications.

Note:
Funding Declaration: This work was supported by the National Key Research and Development Program of China (No. 2022YFC2603800), the National Natural Science Foundation of China (No. 82122042, 82372352, 82030066), Chongqing Science Fund for Distinguished Young Scholars (No. CSTB2022NSCQ-JQX0007), Chongqing Medical Scientific Research Project (No. 2023ZDXM021, 2023QNXM001).

Conflict of Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical Approval: Human serum samples were supplied and approved by Southwest Hospital in Chongqing, China (Approval No. (A)KY2023117).

Keywords: sandwich-type electrochemical aptasensor, exosome detection, rolling circle amplification (RCA), DNA four-way junction (4-WJ), G-quadruplex