Platinum-based Nanozyme screening coupled with ISAR-generated pyrophosphate for dual-mode detection of cardiovascular disease-associated miRNA-21

Early assessment of cardiovascular disease (CVD)-associated molecular alterations may provide valuable information for disease monitoring and clinical evaluation. MicroRNA-21 (miRNA-21) is involved in multiple cardiovascular pathological processes and has been widely investigated as a CVD-associated circulating biomarker. Rationally modulating the activity of platinum-based nanozymes can expand sensing strategies and improve the performance of miRNA-21 detection. Herein, four platinum-based nanozymes are synthesized via a one-step reduction method and systematically evaluate their peroxidase (POD)-like catalytic activities. Among them, the NiPt nanozyme exhibits a favorable apparent peroxidase-like activity under an equal mass concentration, with the lowest Michaelis-Menten constant of 0.462 mM. Then, a colorimetric and fluorescent dual-mode biosensor is developed for the rapid and sensitive detection of miRNA-21 via NiPt nanozyme couple with the target-triggered invading stacking primer-based amplification reaction (ISAR). Sequence-specific hybridization between miRNA-21 and the designed hairpin DNA probe initiates ISAR and generates pyrophosphate (PPi), which suppresses the peroxidase-like activity of NiPt NPs and enables fast, instrument-free, and colorimetric qualitative detection of miRNA-21 at 5 × 10^-11 M. Simultaneously, the Cy5 fluorescence signal exhibits sensitive quantitative detection of miRNA-21 with a limit of detection (LOD) of 2.27 × 10^-13 M over a linear range of 10^-12-10^-9 M (R² = 0.971). The biosensor demonstrated high selectivity toward miRNA-21 against representative interfering miRNA sequences and showed consistency with qRT-PCR in clinical serum samples from heart failure patients and healthy individuals. This dual-mode platform provides a rapid, sensitive, and reliable approach for miRNA-21 detection, demonstrating potential utility for CVD-associated biomarker analysis and further clinical investigation.