We have characterized thiol-derivatized, single-stranded DNA
(5‘-HS-(CH2)6-CAC GAC GTT GTA AAA
CGA CGG CCA G-3‘, abbreviated HS-ssDNA) attached to gold via a
sulfur−gold linkage using X-ray photoelectron
spectroscopy (XPS), ellipsometry, and 32P-radiolabeling
experiments. We found that hybridization of
surface-bound
HS-ssDNA is dependent on surface coverage. The buffer
concentration of the HS-ssDNA solution was found to
have a profound effect on surface coverage, with adsorption greatly
reduced at low salt concentration. More precise
control over surface coverage was achieved by creating mixed monolayers
of the thiol-derivatized probe and a spacer
thiol, mercaptohexanol (MCH), by way of a two-step method, where first
the gold substrate is exposed to a micromolar
solution of HS-ssDNA, followed by exposure to a millimolar solution of
MCH. A primary advantage of using this
two-step process to form HS-ssDNA/MCH mixed monolayers is that
nonspecifically adsorbed DNA is largely removed
from the surface. Thus, the majority of surface-bound probes are
accessible for specific hybridization with
complementary oligonucleotides and are able to discriminate between
complementary and noncomplementary target
molecules. Moreover, the probe-modified surfaces were found to be
stable, and hybridization reactions were found
to be completely reversible and specific in a series of experiments
where duplex melting was examined.
We have developed an electrochemical method to quantify the surface density of DNA immobilized on gold. The surface density of DNA, more specifically the number of nucleotide phosphate residues, is calculated from the amount of cationic redox marker measured at the electrode surface. DNA was immobilized on gold by forming mixed monolayers of thiol-derivitized, single-stranded oligonucleotide and 6-mercapto-1-hexanol. The saturated amount of charge-compensation redox marker in the DNA monolayer, determined using chronocoulometry, is directly proportional to the number of phosphate residues and thereby the surface density of DNA. This method permits quantitative determination of both single- and double-stranded DNA at electrodes. Surface densities of single-stranded DNA were precisely varied in the range of (1-10) x 10(12) molecules/cm2, as determined by the electrochemical method, using mixed monolayers. We measured the hybridization efficiency of immobilized single-stranded DNA to complementary strands as a function of the immobilized DNA surface density and found that it exhibits a maximum with increasing surface density.
Neutron reflectivity was used to determine the concentration profiles of oligomeric DNA monolayers
on gold in high salt concentrations (1 M NaCl). These monolayers are of interest as models for DNA probe
systems used in diagnostic devices. To facilitate its attachment, the DNA was functionalized at the 5‘ end
with a thiol group connected to the oligonucleotide by a hexamethylene linker. Concentration profiles determined
from neutron reflectivity indicate that adsorbed layers of single-stranded DNA (HS-ssDNA) on bare gold are
compact, suggesting the presence of multiple contacts between each DNA strand and the surface. After treatment
with mercaptohexanol, a short alkanethiol with a terminal hydroxy group, the DNA “stands up” and extends
farther into the solvent phase. These changes are consistent with the DNA remaining attached through its
thiol end group while contacts between DNA backbones and the surface are prevented by the formation of a
mercaptohexanol monolayer. The end-tethered HS-ssDNA layer readily hybridized to its complementary
sequence, resulting in DNA helices with a preferred orientation toward the substrate normal.
Current interest in melanin films derived from the autoxidation of dopamine stems from their use as a universal adhesion layer. Here we report chemical and physical characterization of polydopamine films deposited on gold surfaces from stirred basic solutions at times ranging from 2 to 60 min, with a focus on times ≤10 min. Data from Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and electrochemical methods suggest the presence of starting (dopamine) and intermediate (C=N-containing tautomers of quinone and indole) species in the polydopamine films at all deposition times. A uniform overlayer analysis of the XPS data indicates that film thickness increased linearly at short deposition times of ≤10 min. At deposition times ≥10 min, the films appeared largely continuous with surface roughness ≈ ≤ 2 nm, as determined by atomic force microscopy (AFM). Pinhole-free films, as determined by anionic redox probe measurements, required deposition times of 60 min or greater.
This report investigates the effect of DNA length and the presence of an anchoring group on the assembly of presynthesized oligonucleotides at a gold surface. The work seeks to advance fundamental insight into issues that impact the structure and behavior of surface-immobilized DNA layers, as in, for instance, DNA microarray and biosensor devices. The present study contrasts immobilization of single-stranded DNA (ssDNA) containing a terminal, 5' hexanethiol anchoring group with that of unfunctionalized oligonucleotides for lengths from 8 to 48 bases. Qualitatively, the results indicate that the thiol anchoring group strongly enhances oligonucleotide immobilization, but that the enhancement is reduced for longer strand lengths. Interestingly, examination of the probe coverage as a function of strand length suggests that adsorbed thiol-ssDNA oligonucleotides shorter than 24 bases tend to organize in end-tethered, highly extended configurations for which the long-term surface coverage is largely independent of oligonucleotide length. For strands longer than 24 bases, the surface coverage begins to decrease notably with probe length. The decrease is consistent with a less ordered arrangement of the DNA chains, presumably reflecting increasingly polymeric behavior.
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