Dual Quantum Dot Molecular FRET Probes for Picomolar DNA Hexaplexing

Semiconductor quantum dots (QD) combine bright and color-tunable photoluminescence (PL) with large surfaces. Thus, merging multiplexed PL detection and specific biorecognition on a single QD is in principle possible. By attaching multiple DNAs per QD and adjusting terbium (Tb) to QD Förster resonance energy transfer (FRET) with sub-nanometer resolution, we designed dual-color FRET-nanoprobes with six distinguishable signals. The hexaplexing molecular probes combined two PL colors with three PL decays while requiring only a single excitation wavelength for rapid and simple biosensing. Implementation into a sensitive diagnostic assay was demonstrated via the specific quantification of six different DNA targets at picomolar concentrations (2 to 40 femtomole) from a single 200 µL sample. The orthogonality of specific biorecognition and FRET multiplexing on the nanosurface makes the QD-based FRET probes extendable to the sensitive quantification of potentially any biomarker or biological interaction at even higher multiplexing capability. Our results demonstrate that luminescent nanoprobes are capable of translating sophisticated multiplexing approaches into relatively simple biosensing methods.