Table 1.

Average height of the fluorescent marker above the surface as measured by fluorescence interference (SSFM) and the optical thickness of the DNA layer on the surface (measured by WL reflectance)

In all experiments we start with a ssDNA covalently bound to the surface (shown on the left in red). In one set of experiments, the first oligonucleotide (either 50- or 21-mer) is covalently bound to the surface and does not have a fluorescent label. In this case, only WL measurements can be performed prior to hybridization. After hybridization with a fluorescently labeled matching strand (shown on the right in blue), SSFM yields the average fluorophore height attached to either proximal or distal end of the resulting dsDNA. In the second set of experiments, covalently bound oligonucleotide (either 50-or 21-mer) is fluorescently labeled at the distal end and SSFM yields the average height of the ssDNA. When a second, unlabeled matching strand is hybridized, the DNA layer now consists of two species: the dsDNA and the unhybridized strands both of which are carrying a fluorescent marker, but expected at different heights. SSFM measures the average height of the fluorophores. Experiments with 50-mers also include when the first strand is annealed with a 21-mer complementary to either its top or bottom part as schematically shown. The values shown are averages from multiple measurements. Whereas the accuracy of a single height measurement is ≈0.2 nm, the variation between different measurements is ≈10% of the nominal values.