Cascade self-priming DNA polymerization initiated primer exchange reaction for label-free microRNA analysis in preeclampsia

Accurate and sensitive detection of microRNAs (miRNAs) is critical for the early diagnosis and management of pregnancy-related disorders such as preeclampsia, yet remains challenging due to their low abundance and high sequence homology. Here, we report a novel label-free miRNA sensing platform that integrates cascade self-priming DNA polymerization with primer exchange reaction (PER) to achieve high specificity and signal amplification. To overcome the susceptibility of conventional PER to off-target priming, we engineered a structured detection probe in which the primer-binding site of the PER template is blocked by a complementary strand, ensuring target-dependent activation. The assay employs two sequential self-priming DNA polymerization steps: the first is triggered by target miRNA hybridization to the sensing probe, and the second occurs on the displaced blocking strand, which ultimately releases the PER template. This cascaded design substantially enhances both specificity and sensitivity. The liberated PER template then initiates autonomous primer extension, generating abundant G-rich repeats that fold into G-quadruplex (G4) structures. These G4 are selectively recognized by thioflavin T (ThT), yielding a turn-on fluorescence signal without the need for covalent labeling. Under optimized conditions, the assay exhibits a broad dynamic range from 1 fM to 100 pM with a detection limit of 0.42 fM, and demonstrates excellent discrimination against single- to triple-base mismatches and unrelated miRNAs. Moreover, the method shows high reproducibility, stability, and accurate recovery in diluted human serum, with results strongly correlating with RT-qPCR. By combining dual self-priming amplification with a label-free PER readout, this work provides a robust, isothermal, and sensitive strategy for miRNA quantification, holding considerable potential for non-invasive clinical diagnostics and early detection of preeclampsia.