Single-Molecule Study of an Adsorbed Oligonucleotide Undergoing Both Lateral Diffusion and Strong Adsorption

Abstract

Single-molecule fluorescence spectroscopy was used to probe the lateral transport of an adsorbed oligonucleotide, the SP6 promoter primer, which is a 24-mer labeled at the 5′ end with tetramethylrhodamine. The oligonucleotide was adsorbed to the interface of an aqueous solution of 0.01 M KCl and silica chemically modified by chlorodimethyloctadecylsilane. Confocal fluorescence microscopy achieved single-molecule resolution, with a molecule in the beam 7% of the time. Autocorrelation of the data fit well to a model having two species, one diffusing and the other one undergoing transient strong adsorption. A small number of bursts, 0.3%, had unusually long durations, accounting for the slow component in the autocorrelation. When these long bursts were excised from the data, the autocorrelation fit well to simple diffusion, with D ) 4 × 10 -6 cm 2 /s. Autocorrelation of the long bursts alone gave a rate constant for desorption of 3 s -1 . The strongly adsorbed molecules were found to comprise 10% of the total population of adsorbates. It is concluded that the lateral transport of the SP6 promoter primer is described by fast lateral diffusion interrupted by rare, reversible, strong adsorption to defect sites. During strong adsorption, the tetramethylrhodamine label undergoes hindered motion, suggesting it is not the adsorbing moiety.

The Vroman Effect: A Molecular Level Description of Fibrinogen Displacement

“…In the somewhat analogous problem of analyte retention on chromatography columns, [119][120][121] the literature indicates that very long retention times on silica can be related to isolated surface silanol groups which afford strong hydrogen bonds. On the other hand, interactions with deprotonated silanols seem to be associated only with shorter retention times.…”

Enhanced Fluorescence of Cy5-Labeled Oligonucleotides Near Silver Island Films: A Distance Effect Study Using Single Molecule Spectroscopy

“…The "off" state with duration of tens to seconds could be associated with a stabilized nonfluorescent Cy5 adsorbed to neighboring biomolecules. 39 The duration of "dim" or "off" state would be most likely to be associated with the time of sticking event. As an additional effect with silvered surfaces, the metallic nanostructure introduces new electromagnetic decay channels to the system, rendering excitons incapable of accepting electrons from the residue and creating a relatively reductive environment.…”

Molecular Approaches to Chromatography Using Single Molecule Spectroscopy

“…Oligonucleotides are known to hydrogen-bond, making RPLC the preferred purification technique. In Wirth and Swinton, 149 the transport of a fluorescently labeled 24-mer over C 18 functionalized silica was investigated by confocal microscopy. FCS analysis showed bimodal diffusion where the diffusion coefficients differed by 2 orders of magnitude ( D 1 = 2 × 10 –8 and D 2 = 2 × 10 –6 cm 2 /s); the two populations were attributed to free diffusion and a distorted slow diffusion value due to adsorption events.…”