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

Previous investigations into DNA orientation under an electrostatic field used either a low-intensity or a pulsed field, and the measurements were made in the region where the degree of orientation was relatively low. This was because applying high-intensity steady fields to aqueous solutions resulted in a temperature rise and caused turbulence or boiling, which interfered with orientation. In this paper, microfabricated electrodes are used to obtain stationary AC electric fields in excess of 1 MV/m. Planar microelectrodes with either 15- or 60-μm gaps, 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 has a high surface-to-volume ratio, Joule heat is efficiently removed, so that a very high-intensity field can be created without excessive temperature rise. A high-sensitivity detection method is required for measurements with microelectrodes, due to the small number of molecules involved. For this purpose, a fluorescent dye is intercalated into the bases of the 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 an index of DNA orientation