Mutually Activated Dual-Exponential Amplification DNA Machine Enhances Robust CRISPR/Cas12a Feedback Propagation for Ultrasensitive miRNA Detection

Abstract

Breast cancer (BC) remains a critical global health challenge, necessitating ultrasensitive methods for detecting biomarkers such as miR-155, a key regulator in BC progression. Here, we present a mutually activated dual-exponential amplification DNA machine (MADEA-DNA machine) for ultrasensitive miR-155 detection. This system integrates exponential rolling circle amplification (E-RCA) and autocatalytic incremental strand displacement amplification (AI-SDA), driven by a bidirectional activation mechanism. Target miR-155 initiates E-RCA via a functional primer probe (FPP) or AI-SDA through a functional hairpin probe (FHP), with amplification products cross-activating the counterpart system to establish a self-reinforcing loop. The resultant amplicons further activate CRISPR/Cas12a, enabling the trans-cleavage of fluorescent reporters for signal amplification. The MADEA-DNA machine achieves a detection limit of 1.26 fM, with a dynamic range spanning 5 fM–10 nM, and demonstrates exceptional specificity against mismatched and nontarget miRNAs. Validation in human serum revealed significantly elevated miR-155 levels in BC patients versus healthy donors, corroborated by qRT-PCR. This system combines machine-like operational efficiency, dual-amplification synergy, and CRISPR-enhanced sensitivity, offering a robust platform for liquid biopsy applications in early BC diagnostics.