Influence of the Thiol Anchor on the Orientation of Surface‐Grafted dsDNA Assemblies

Abstract

The orientation of surface-grafted dsDNA assemblies relative to the surface depends strongly on the nature of the employed thiol anchor. This was shown by ssDNA capture probe strands of 20 bases grafted to a gold surface by three dithiane rings or a single mercaptohexyl group. The capture probe strands were hybridized to one end of complementary ssDNA strands (target) comprising 40, 60, or 80 bases (T , T , and T ). At the other end of the targets a fluorophore-labeled reporter probe ssDNA strand of 20 bases was hybridized. To stiffen the DNA assemblies, the targets T and T were further hybridized to ssDNA patches of 20 or 40 bases. Whether the fluorescence intensity, and thus the distance between surface and fluorophore, increases or decreases with increasing target length depends on the thiol anchor. Attempts were made to heal the nicks that are present in the formed dsDNA assemblies by ligation. For enzymatic ligation, the presence of a phosphate at the 5'-end of the reporter probe and a patch is required, which may also influence the fluorescence intensity.

Langmuir Analysis of the Binding Affinity and Kinetics for Surface Tethered Duplex DNA and a Ligand–Apoprotein Complex

“…However, as is obvious from Figure , we could not observe significant unbinding of complementary DNA upon rinsing with buffer. This finding is in good agreement with previous studies by us and other groups. ,,− ,, …”

“…9,23 Also when we monitored DNA hybridization at surfaces by SPR, SPFS, or QCM, we were not able to release a significant amount of target molecules by rinsing with buffer solution. 7,24,25 Based on our results, we began to question whether DNA hybridization at surfaces can be generally and unequivocally described by the Langmuir model. If in these experiments DNA hybridization/dehybridization can be described by the Langmuir model, the value for k off has to be very low.…”

“…surface plasmon resonance (SPR), , surface plasmon fluorescence spectroscopy (SPFS), , or ellipsometry, or by quartz crystal microbalance (QCM) measurements as an acoustic method. − Besides direct attachment of the capture probe to a gold surface also the streptavidin–biotin system has been widely employed. , For this purpose, first biotin-terminated tethers are chemisorbed to gold via thiol anchors and employed to assemble a streptavidin layer at the surface. , In this case, up to two binding sites of the homotetrameric streptavidin were used for surface attachment, and two binding sites remain, which can be used to bind biotinylated ssDNA capture probe strands in a sandwich type assembly. As an advantage of this method the capture probe strands are more evenly distributed over the surface, and the probability for direct interaction between two capture probe strands can be minimized. , Besides gold surfaces also glass surfaces have been modified with ssDNA capture probe strands covalently. , For many biosensing applications, it is assumed that DNA hybridization at the surfaces follows, at least partially, the Langmuir model of adsorption. ,− However, in only a few papers, distinct experiments were carried out to prove this assumption. ,, When we developed surface bound molecular beacons (MBs) in order to trace specific oligonucleotide sequences at surfaces via SPFS, we recognized that after hybridization with the target almost none of the target molecules can be released by intense rinsing with the same buffer solution that was used also during the hybridization event. , Also when we monitored DNA hybridization at surfaces by SPR, SPFS, or QCM, we were not able to release a significant amount of target molecules by rinsing with buffer solution. ,, Based on our results, we began to question whether DNA hybridization at surfaces can be generally and unequivocally described by the Langmuir model. If in these experiments DNA hybridization/dehybridization can be described by the Langmuir model, the value for k off has to be very low.…”

Surface Plasmon-Assisted Fluorescence Enhancing and Quenching: From Theory to Application

“…Moreover, the DNA monolayer overlying the gold surface has been further characterized in terms of the areal number density and thiol-anchor support. − The orientation of surface-grafted double-stranded DNA assemblies relative to the gold surface is mainly defined by the nature of the employed thiol anchor . When the tilt angle of the local double helix is fixed to the surface, the thiol anchor plays a large role in scaling the spatial separation distance of the fluorophore to a metallic surface.…”

“…69−71 The orientation of surface-grafted double-stranded DNA assemblies relative to the gold surface is mainly defined by the nature of the employed thiol anchor. 72 When the tilt angle of the local double helix is fixed to the surface, the thiol anchor plays a large role in scaling the spatial separation distance of the fluorophore to a metallic surface. And the areal number density of DNA on the gold surface actually determines the overall stiffness of the DNA matrix.…”