11 research outputs found
Controlled and Efficient Hybridization Achieved with DNA Probes Immobilized Solely through Preferential DNA-Substrate Interactions
Quantitatively correlating the response of DNA sensors to clinically and biologically significant sample abundances requires optimizing the reproducibility of hybridization between DNA targets and surface-bound DNA probes. 1,2 Although gold surfaces are not used in commercial fluorescence-based DNA arrays, gold offers many useful properties as a model substrate 3 and has been successfully used in systematic studies of the interactions governing DNA hybridization near interfaces. 4-8 The hybridization behavior of surface-immobilized DNA probes strongly depends on the probe conformation and on the lateral spacing between adjacent probes. Hybridization is generally enhanced for DNA probes that extend away from a surface, in part because steric hindrance is lower for such brushlike probes than for DNA directly adsorbed on gold. At surface densities e5 × 10 12 cm -2 , typical probe sequences (15-30 nucleotides) can hybridize with efficiencies >60%, 7-10 but at probe densities >1 × 10 13 cm -2 hybridization efficiency is often reduced because of increased electrostatic repulsion and steric constraints. 11-13 Surfaces with DNA probes in upright conformations can be prepared following a number of strategies, including adsorption via a thiol incorporated at one end (DNA-SH) or coupling DNA probes to bifunctional monolayers. [4][5][6][7][8][14][15][16][17][18] Preparing DNA probes * To whom correspondence should be addressed. E-mail: opdahl
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Sum frequency generation (SFG) surface vibrational spectroscopy and atomic force microscopy (AFM) studies of the composition, structure, and mechanical behavior of polymers at interfaces
Influence of Attachment Strategy on the Thermal Stability of Hybridized DNA on Gold Surfaces
The thermal stabilities of double-stranded
DNA hybrids immobilized
on gold surfaces are shown to be significantly affected by the conformation
of the hybrid. To analyze this behavior, DNA probes were immobilized
using attachment strategies where the nucleotides within the strand
had varying levels of interactions with the gold substrate. The abilities
of these probes to form double-stranded hybrids with solution DNA
targets were evaluated by surface plasmon resonance (SPR) over a temperature
range 25–60 °C. The measurements were used to construct
thermal stability profiles for hybrids in each conformation. We observe
that DNA hybrids formed with probe strands that interact extensively
with the gold surface have stability profiles that are shifted lower
by 5–10 °C compared to hybrids formed with end-tethered
probes that have fewer interactions with the surface. The results
provide an understanding of the experimental conditions in which these
weaker DNA hybrids can form and show the additional complexity of
evaluating denaturation profiles generated from DNA on surfaces
Temperature Gradient Approach for Rapidly Assessing Sensor Binding Kinetics and Thermodynamics
We report a highly resolved approach
for quantitatively measuring
the temperature dependence of molecular binding in a sensor format.
The method is based on surface plasmon resonance (SPR) imaging measurements
made across a spatial temperature gradient. Simultaneous recording
of sensor response over the range of temperatures spanned by the gradient
avoids many of the complications that arise in the analysis of SPR
measurements where temperature is varied. In addition to simplifying
quantitative analysis of binding interactions, the method allows the
temperature dependence of binding to be monitored as a function of
time, and provides a straightforward route for calibrating how temperature
varies across the gradient. Using DNA hybridization as an example,
we show how the gradient approach can be used to measure the temperature
dependence of binding kinetics and thermodynamics (e.g., melt/denaturation
profile) in a single experiment
Effect of Probe–Probe Distance on the Stability of DNA Hybrids on Surfaces
We
have used temperature gradient surface plasmon resonance (SPR)
measurements to quantitatively evaluate how the stability of different
types of hybrids formed with DNA probes on surfaces is affected by
probe spacing. SPR sensors with different average surface densities
of probes were prepared by coadsorbing probes with lateral spacers
strands comprised of phosphorothioated adenine nucleotides (A15*).
Increasing the fraction of A15* spacers in the immobilization solution
results in larger distances between probes on the sensor, determined
here using a combination of SPR and X-ray photoelectron spectroscopy
(XPS) measurements. The hybridization activities of probes were simultaneously
measured over a temperature range that spanned the denaturation temperature
(<i>T</i><sub>m</sub>) of hybrids by applying a spatial
temperature gradient across the sensor surface. The resulting temperature
profiles of hybridization activity show how the stability of hybrids
increases as either the distance between probes or the ionic strength
of the hybridization buffer increase. Additionally, hybridization
activity profiles sharpen as the spacing between probes increases,
indicating more homogeneous hybridization behavior of probes. The
results provide quantitative experimental data for testing theoretical
models of stability, supporting models that account for both repulsive
interactions between DNA strands and local variability in probe surface
density
Synthesis and structural characterization of glucopyranosylamide films on gold
Self-assembled monolayers (SAMs) of glucose derivatives on gold have been prepared from α- and β-glucopyranosylamide derivatives. The glucosyl conjugates were synthesized stereoselectively via the in situ generation of glucosyl isoxazolines followed by treatment with thiopyridyl esters. The resulting film structures were characterized by atomic force microscopy, reflection Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. The experimental data indicated that α- or β-linked glucopyranosylamide derivatives with free hydroxyl groups attach to gold via the thiol linker. Both derivatives form monolayer films with high packing densities-comparable to those typically observed for alkanethiol monolayers on gold. Acetate analogues of these conjugates do not form SAMs on gold; they form multilayered films under identical deposition conditions. © 2007 American Chemical Society