Quantitative analysis of DNA orientation in stationary AC electric field using fluorescence anisotropy

In this study, micro-fabricated electrodes are used to obtain stationary field in excess of 1 MV/m. Micro-electrodes with either 15 μm or 60 μm gap, depending on the length of sample DNA to be used, are fabricated with planar technique on glass substrates. Because the high-field region in the gap is small and having high surface to volume ratio, Joule heat is efficiently removed, so that very high intensity field can be created without excessive temperature rise. A high-sensitive detection method is required for the measurements with micro-electrodes, due to a small number of molecules involved. For this purpose, a fluorescent dye is intercalated into the bases of DNA, and the optical polarization of emitted fluorescence is measured. The polarization components of the emitted light, both parallel and perpendicular to the applied electrostatic field, are measured independently, and fluorescent anisotropy, the ratio of (the difference between parallel and perpendicular polarization) to (total emission) is used as the index of DNA orientation. Field-intensity dependence of fluorescence anisotropy is measured using pUC18 (2.7 kb DNA), and the result is compared with an analytical model. The measured polarization factor is found to be several orders of magnitude larger than that of a conducting ellipsoid with the same dimension. This can be explained by assuming a `swelling´ of electrical equivalent diameter of DNA by 20 nm, comparable to the Debye length, i.e. the thickness of counter ion cloud. The amount of counter ion is varied by changing pH of the medium, keeping its conductivity constant. As pH increases, increase in the anisotropy is observed, in particular between pH 5 and 6. This is attributed to the dissociation of phosphate groups, by which negative charge density on the DNA backbone is increased. Multivalent cations, Ca ²⁺, Mg²⁺, Zn²⁺ and Al³⁺, are expected to bind to DNA backbone and reduce polarization. However, no significant change of anisotropy is observed in the concentration range of 10^-4-10^-6 in our experimental conditions. Further increase in concentration was prevented by conductivity increase