Construction of a Bifunctional Single-Quantum Dot Nanosensor for Enzyme-Free Detection of N ⁶ -Methyladenosine Demethylase in Breast Cancer

In mammalian organisms, <i>N</i>⁶-methyladenosine (m6A) serves as the most prominent internal modification of mRNA, and it critically regulates gene expression and cancer progression through dynamic erasure by demethylases (e.g., Fat Mass and Obesity-associated protein (FTO)). As an oncogenic m6A "eraser", FTO promotes breast cancer development via suppression of apoptosis and metabolic reprogramming. Herein, we develop a bifunctional single-quantum dot (QD) nanosensor for an FTO assay by integrating m6A-responsive DNAzyme with enzyme-free signal amplification and single-molecule detection. In this nanosensor, the 605QD serves as both a scaffold for constructing a streptavidin-anchored DNA walker assembly and a donor for efficient Förster resonance energy transfer (FRET). When FTO is present, the m6A-locked 8-17E DNAzyme is activated to initiate the 3D DNA walker-mediated cleavage of hairpin probes on the 605QD surface, liberating the binding sites for dual Cy5-labeled signal probes. Subsequently, the fractured hairpin probe and the signal probe form a three-way junction (TWJ) skeleton on the 605QD surface, generating a distinct FRET signal. This nanosensor enables simple, rapid, and enzyme-free detection of FTO and achieves a remarkably low limit of detection (LOD) of 41.7 fM within 90 min. Moreover, it can be applied for the detection of endogenous FTO in a single cell, kinetic analysis, inhibitor screening, and discrimination of the FTO level in breast cancer tissues and healthy counterparts. With the advantages of rapid analysis, minimal sample requirements, and clinical-grade precision, this single 605QD-based nanosensor provides a new paradigm for breast cancer epigenetic research and point-of-care diagnostics.