Abstract
Abstract The dynamical behavior of DNA, which was immobilized on DNA microarray, have puzzled the gene chip researchers. In general, there are two types of immobilization methods, i.e. charge-charge interaction by poly-L-lysine coating and covalent binding to the glass surface at the one end of DNA. In this study, we aim to distinguish that either DNA is like a seaweed wavering in the solution (wavering mode) or like a rope sticking on the surface (sticking mode) of glass. The two modes have quite different hybridization efficiency. Because evanescent wave generated by TIRFM exhibits exponential decay with increasing distance z from the interface, the signal intensity excited by evanescent wave is thus sensitive to the change of the location of the signal emmitor along z direction. Therefore, the TIRFM is adequate to gauge the vertical location of the fluorescence dye. We measure the TIRF signal of Cy3 labeled 20-bp DNA molecules which was immobilized on the coverslip via a biotin-streptavidin. To determine which mode that the behavior DNA are belong to,,we record the time trace of the single Cy3 fluorescence intensity to see if there is a changes in intensity corresponding to the vertical displacement. We also performed the same measurement for the other type of immobilization methods, i.e. charge-charge interaction by poly-L-lysine coating. In this study, we will also report the theoriotical estimated size of fluorescence signal produced by the wavering of dye in the exponentially decayed evanescent excitation field. The amount and the time characteristic of the wavering is determined by Brownian motion. This wavering signal should be larger enough to stand out from the noise of a steaty single dye.