In Situ Phosphate-Directed Growth of Dual-Emissive Lanthanide MOFs on DNA for Amplification-Free Ratiometric Epigenetic Sensing

Abstract The ultrasensitive detection of epigenetic DNA modifications typically relies on enzyme-dependent amplification, which is often hindered by structural bias and matrix interference. Herein, we report a programmable, amplification-free ratiometric sensing platform driven by in situ growth of dual-emissive lanthanide metal−organic frameworks (Ce/Eu-MOFs) directly onto nucleic acid templates. Mechanistic investigations, integrating 31P NMR spectroscopy and density functional theory (DFT) calculations, reveal that the DNA phosphate backbone acts as the thermodynamically favored nucleation site, governing the regioselective assembly of the Ce/Eu-MOFs. Furthermore, the integration of computational and experimental approaches established a rational screening principle to identify dipicolinic acid as the optimal MOF ligand. By coupling this in situ MOF growth with selective chemical labeling and magnetic enrichment, the designed scaffold enables highly specific recognition and self-calibrating ratiometric quantification of 5-formylcytosine (5fC), a critical epigenetic biomarker. This platform achieves exceptional amplification-free sensitivity with a limit of detection of 0.001%, demonstrating strong quantitative agreement with the clinical gold-standard ELISA-based analysis. Finally, the proposed method was successfully applied to profile dynamic 5fC variations across complex tissue samples. This work establishes a biomolecule-directed Ln-MOF growth strategy for precise epigenetic analysis, providing a versatile and robust framework for developing advanced, amplification-free analytical tools.