Improved Thermal Stability and Homogeneity of Low Probe Density DNA SAMs Using Potential-Assisted Thiol-Exchange Assembly Methods

Abstract

Methods for producing DNA SAM-based sensors with improved thermal stability and control over the homogeneity of low DNA probe density will enable advanced sensor development. The thermal stability of low-coverage DNA SAMs was studied for surfaces prepared using potential-assisted thiol exchange (E dep ) and compared to DNA SAMs prepared without control over the substrate potential (OCP dep ). Both surface preparation methods were studied using in situ fluorescence microscopy and electrochemistry with fluorophore or redox-modified DNA SAMs on a single-crystal gold bead electrode. Fluorescence microscopy showed that the influence of the underlying surface crystallography was important in both cases. The highest thermal stability was realized for square or rectangular surface atomic structure (e.g., surfaces from 110 to 100). The 111 and related surfaces were the least thermally stable. The low DNA coverage surfaces prepared by E dep had better thermal stability and higher DNA probe mobility as compared to OCP dep -prepared surfaces with the similar coverage. These results were correlated with methylene blue redox-tagged DNA probes, which directly measured the average DNA coverage. Both methods indicated that E dep DNA SAMs were more uniformly distributed across the electrode surface, while the surfaces prepared via OCP dep assembled into clusters with reduced mobility. The potentialassisted thiol-exchange approach to preparing low-coverage DNA SAMs was shown to quickly create modified surfaces that were consistent, had mobility characteristics which should yield superior DNA hybridization efficiencies, and having greater thermal stability which will translate into a longer shelf-life.

Signal Amplification in Electrochemical DNA Biosensors Using Target-Capturing DNA Origami Tiles

“…Despite this, target hybridization efficiencies are still high and even suggested unexpectedly to be supported by regions of dense probe clustering . Emerging methods for controlling the distance of neighboring immobilized probes and the incidence of clustering on gold electrodes through electrodeposition are prevalent in the literature. − However, as no method of controlling the specific confirmation of the probe spacing has been employed here, the particular degree of uniformity in probe spacing cannot be confirmed. This would be expected to produce a degree of variability in the electrochemical characterization of the functionalized electrode condition.…”

Redox-Controlled Energy Transfer Quenching of Fluorophore-Labeled DNA SAMs Enables In Situ Study of These Complex Electrochemical Interfaces

“…Either Alexafluor488 (AF488) or Methylene blue (MB) labels labeled the 3′ end of the oligo. The DNA oligos were pretreated as described in the Supporting Information (SI) . All DNA stock solutions were used within 2 weeks after reduction of the disulfide.…”

“…The fluorescence images of the DNA SAMs were analyzed as described previously . Each fluorescence image was analyzed after removing the background.…”

“…The fluorescence images of the DNA SAMs were analyzed as described previously. 28 Each fluorescence image was analyzed after removing the background. The different crystallographic regions on the single-crystal bead electrode were selected as circular regions of interest (ROI) with a 16 pixel or 50 μm diameter.…”

“…The DNA oligos were pretreated as described in the Supporting Information (SI). 28 All DNA stock solutions were used within 2 weeks after reduction of the disulfide. The DNA working solution (1 μM) was made by diluting the DNA stock with an immobilization buffer (IB) composed of 10 mM TRIS (2-amino-2-(hydroxymethyl)propane-1,3-diol) buffer containing TRIS Base and TRIS HCl (Bioperformance 99.0%, Sigma-Aldrich), 100 mM NaCl (99.5% BioXtra, Sigma-Aldrich), and 25 mM MgCl 2 (99% Sigma-Aldrich) adjusted to pH 7.5.…”