Quantum-dot-based multiplexed fluorescence resonance energy transfer

Abstract

Colloidal semiconductor quantum dots (QDs) have narrow photoemission bandwidths and broad absorption spectra that are ideal for multiplexing applications. In contrast to organic dyes, which require a complex arrangement of excitation sources and filters to generate multiple signals, many populations of QDs can be simultaneously excited with a single excitation source. In a mixed sample, the narrow and symmetric emission profile of QDs allows simple deconvolution of the composite signal to generate individual QD photoluminescence (PL) contributions. We have shown that CdSe-ZnS core-shell QDs function as efficient energy donors in fluorescence resonance energy transfer (FRET) systems. In this study, we tested several QD-protein bioconjugates, each having a unique PL spectrum (or "color") functioning as independent signal channels, to assess the feasibility of a QD FRET-based multiplexing system. Several populations of QDs were self-assembled with labeled and unlabeled proteins, mixed in solution and excited at single wavelength. The resulting spectra were deconvoluted using the known QD emission profiles to reveal individual contributions of each QD population. QDs coated with dye-labeled protein acceptors showed distinct FRET-induced PL quenching due to the presence of proximal dye acceptors. Steady-state fluorescence results were verified by time-resolved spectroscopic data from the mixed samples where a reduced QD lifetime indicated the presence of proximal dye quencher on one or more QD populations. We will discuss how these findings are used to develop QD-based FRET multiplexed biosensors using a similar strategy where each QD population has surface-bound proteins that are sensitive to a unique molecular target.