Intelligent molecular cleavage and dual-signal relay amplification ratiometric strategy for high-sensitivity analysis and dynamic monitoring of exosomal RNA in glioma

Abstract

Exosomal RNA has emerged as a promising biomarker for glioblastoma (GBM) due to its exceptional stability in biofluids and strong correlation with tumor progression. In this study, we present an innovative intelligent molecular cleavage and dual-signal relay amplification-based ratiometric (ISR) strategy for high-sensitivity monitoring and dynamic analysis of exosomal RNA in glioma. The core mechanism is based on a hollow duplex structure that effectively prevents premature cleavage by duplex-specific nuclease (DSN), ensuring both the accuracy and stability of the detection system. Upon the introduction of the target microRNA (miRNA), one strand of the hollow duplex is displaced, forming a miRNA-DNA duplex that serves as a substrate for DSN, initiating target recycling and signal amplification. This dynamic process, coupled with dual-signal relay amplification, significantly enhances both sensitivity and stability, even at low miRNA concentrations. Our ratiometric approach substantially improves detection accuracy by comparing dual signal outputs. We further demonstrate the capability of real-time tracking of exosomal RNA dynamics, enabling precise monitoring of miRNA fluctuations over time. The practical applicability of our ISR strategy was validated by accurately detecting exosomal miRNA levels in clinical serum samples from glioblastoma patients, distinguishing them from healthy controls with high precision. Our method represents a significant advancement in early cancer detection and disease monitoring, with broad implications for precision medicine and the development of point-of-care diagnostic tools. Looking ahead, the ISR strategy holds great potential for monitoring a wide range of diseases, offering new opportunities for personalized diagnostics and therapeutic strategies.