Fast Fluorescence Titration Quantification of Plasmid DNA with DNA Attractive Magnetic Nanoparticles.

Abstract

Fluorescence titration using magnetic nanoparticles (FTMN) was performed as a rapid, inexpensive, and simple method for quantifying the amount of fluorophore-intercalated plasmid DNA on these DNA attractive nanoparticles. Binding of the propidium iodide (PI)-intercalated DNA (PI/DNA) to polyethylenimine (PEI)-coated monodisperse iron oxide magnetic nanoparticles (PEI-MNs) was confirmed with transmission electron microscopy after the two species were mixed in water for less than a minute. The amount of DNA on PEI-MNs in aqueous solution, however, could not be easily determined using direct fluorescence measurements due to strong scattering by aggregated MNs, especially at high nanoparticle concentrations. Instead, fluorescence measurements were taken immediately after the solution of PI/DNA and PEI-MN mixtures was treated with a magnet to pull the PEI-MNs out of the solution. The detected fluorescence signal of the remaining free PI/DNA in the solution decreased as the concentration of PEI-MNs in the pre-treated solutions increased, resulting in a titration curve, which was used to determine the amount of DNA on MNs, the dissociation constant, and binding energy after the concentration of PEI-MNs was calibrated with microwave-plasma atomic emission spectroscopy. Quantitative polymerase chain reaction was used to understand the binding of DNA to MNs and to measure the amount of free PI/DNA in solution, and the results were similar to those obtained with the FTMN method.