A positively charged QDs-based FRET probe for micrococcal nuclease detection

Abstract

A new strategy for quantitatively detecting micrococcal nuclease (MNase) is proposed using electrostatic interaction-based fluorescence resonance energy transfer (FRET) between positively charged QDs and negatively charged dye-labeled single-stranded DNA (dye-ssDNA). Herein, we have made our attempt to develop a strategy where the variation of FRET efficiency is due to the change of the electrostatic interaction between QDs and the ssDNA that result from the cleavage of dye-ssDNA by a single-strand-specific nuclease. To demonstrate the feasibility of this design, positively charged QDs (lysozyme modified QDs, Lyz-QDs) are prepared as the energy donor, with the fluorescent dye 6-carboxy-X-rhodamine (ROX) that is labeled to ssDNA serving as the energy acceptor. The ROX-labeled probe ssDNA (ROX-ssDNA) is absorbed to the surface of the QDs through electrostatic interaction, which results in resonance energy transfer between the QDs and the dye. In the presence of MNase which cleaves the ROX-ssDNA into small fragments, the weakened interaction between QDs and the shortened ssDNA causes the decrease of FRET efficiency. At given amounts of donor and acceptor, the ratio of fluorescence intensity of QDs to ROX changes in a MNase concentration-dependent manner. Under optimized conditions, the ratio is linear with MNase concentration over the range of 8 × 10⁻³ to 9.0 × 10⁻² unit mL⁻¹, with a limit of detection of 1.6 × 10⁻³ unit mL⁻¹. This new detection strategy features straightforward design and easy operation, which is capable of expanding the application of the positively charged QDs-based FRET in DNA-related bioassays.