All-in-One Nanoflare Biosensor Combined with Catalyzed Hairpin Assembly Amplification for in Situ and Sensitive Exosomal Mirna Detection and Cancer Classification

Abstract

Exosomal miRNAs can reflect tumor progression and metastasis, and are effective biomarkers for cancer diagnosis. However, the accuracy of exosomal miRNA-based cancer diagnosis is limited by the low sensitivity and complicated RNA extraction of traditional approaches. Herein, a novel biosensor is developed for in situ, extraction-free, and highly sensitive analysis of exosomal miRNAs via nanoflare combined with catalyzed hairpin assembly (CHA) amplification. Without cumbersome and costly miRNA extraction or transfection agents, nanoflare can directly enter the exosomes to bind target miRNAs and generate a fluorescence signal that can be amplified by the CHA reaction to achieve the in situ and highly sensitive detection of exosomal miRNAs. Under the optimal conditions, the detection limit of 0.005 fM is obtained for three exosomal miRNAs, which is an order of magnitude lower than quantitative real time polymerase chain reaction (qRT-PCR). In combination with the linear discriminant analysis algorithm, five exosomes are distinguished with 100% accuracy. Importantly, five cancers including breast, lung, liver, cervical, and colon cancer from 64 patients are distinguished with 99% accuracy by testing exosomal miRNAs in clinical plasma. This simple, accurate, and sensitive biosensor holds the potential to be expanded into clinical non-invasive cancer diagnostic tests.

Note:
Funding declaration: This work is financially supported by the National Natural Science Foundation of China (21922402), Liaoning Provincial Natural Science Foundation of China (2021-MS-099) and the Fundamental Research Funds for the Central Universities (N2205004).

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: The study was performed in compliance with the Declaration of Helsinki, and approved by the Life Sciences Ethics Review Committee of Northeastern University (China).

Keywords: Exosomal miRNAs, in situ, Nanoflare, Catalyzed hairpin assembly, Cancer classification