Development of a single quantum dot-mediated FRET biosensor for amplification-free detection of ten-eleven...

Ten-eleven translocation (TET) family proteins play key roles in multiple cellular processes by mediating the oxidation of 5-methylcytosine to directly participate in DNA demethylation, and often aberrantly expressed in various diseases. In this research, we develop a single-quantum-dot (QD)-mediated fluorescence resonance energy transfer (FRET) biosensor for amplification-free measurement of ten-eleven translocation 2 (TET2). When TET2 is present, it catalyzes the oxidation of 5-vinylcytosine in dsDNA to 5-formylmethylcytosine, and the subsequent labeling of dsDNA with Cy5 generates a biotinylated Cy5-dsDNA complex. Biotinylated Cy5-dsDNA complexes are conjugated to the streptavidin-coated 605QDs to obtain a Cy5-dsDNA-605QD nanostructures, inducing FRET from 605QD to Cy5. FRET signal can be simply measured by single-molecule counting. This biosensor enables homogeneous detection of TET2 with a limit of detection (LOD) of 0.042 ng/μL, and it can accurately measure cellular TET2 down to 1 cell. Moreover, this biosensor can be used to screen TET2 inhibitors, offering a new platform for TET2-related medical research and clinical diagnostics.