The studies in the Process Sensing Group are currently focussed on the surface characterization of thiol-derivatized, ssDNA monolayers immobilized on gold surfaces. The coverage, surface structure, hybridization activity of the surface-bound DNA probes are characterized
with a variety of surface-sensitive methods, including x-ray photoelectron spectroscopy (XPS), electrochemical methods, secondary ion mass spectrometry, neutron reflectivity, grazing angle FT-IR, surface plasmon resonance,
ellipsometry, surface-enhanced Raman spectroscopy, and 32P radiolabeling.
The monolayers are formed by a two-step process. In the first step, a piece of clean, bare gold is immersed in a 1 micromolar solution of thiolated, single-stranded DNA (HS-ssDNA).
The HS-ssDNA - coated surface is then exposed to a millimolar solution of a second thiol molecule, mercaptohexanol (MCH).
MCH not only passivates the surface, preventing non-speific adsorption of DNA from solution, but also displaces non-specifically adsorbed HS-ssDNA (those molecules that interact with the surface through some
functionality other than the thiol group).
Hybridization, or pairing of single strands of DNA to form double-stranded DNA, is carried out by exposing the surface immobilized DNA molecules (the 'probes') to a solution containing
single-stranded DNA wiht the complemenary sequence (the 'target').
XPS | ||
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 | We confirmed that the MCH posttreatment step removes non-specifically adsorbed DNA using x-ray photoelectron spectroscopy (XPS). We found that the N 1s peak area in the XPS spectrum is proportional to the amount of DNA adsorbed on the surface. Shown on the left are the N 1s spectra obtained from thiolated and non-thiolated ssDNA before (red lines) and after (blue lines) MCH posttreatment. Posttreatment results in displacement of nearly all the nonthiolated ssDNA. For HS-ssDNA, only a small amount is removed. Clearly, thethiol end plays an important role in anchoring the HS-ssDNA molecule to the surface. Futhermore, the MCH serves to displace DNA molecules that are not adsorbed throuhg the thiol end group. | |
Neutron Reflectivity | ||
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| Neutron reflectivity (NR) was used to determine the volume fraction profile of DNA in-situ with sub-nm resolution. The interface between the substrate and aqueous buffer is located at z = 0, and the DNA layer extends into the buffer in the positive z-direction. The initially adsorbed single-stranded, 25 base HS-ssDNA molecules (black solid line) extend ~ 2.5 nm from the substrate into the buffer. The narrow width of the DNA layer is consistent with the DNA adsorbing to the surface in "flattened" configurations in which several adsorption contacts tend to exist between a DNA molecule and the surface, as schematically illustrated below the graph. After posttreatment with mercaptohexanol, the DNA "stands up" and its width increases to ~ 5 nm (red dashed line), consistent with a primary attachment through the thiol endgroups. After hybridization, the now rigid DNA double helices extend about 7 nm from the substrate (blue dotted line). Comparing this number with the contour length of a 25-mer DNA double helix (8.4 nm) indicates that the double helices adopt an average tilt of about 30 degrees from the substrate normal (surface density of DNA strands: ~ 4 x 1012 chains/cm2). Within experimental precision, 100 % hybridization is observed. |  | |
We monitored hybridization efficiency of the surface-bound probe as a function of HS-ssDNA coverage using 32P radiolabeling.
A series of samples with varying coverages were prepared. These substrates were exposed to 32P-labeled complementary DNA.
A substrate with only MCH was also included in these experiments as a control. In the image shown to left, the amount of surface-bound DNA increases from right to left.
The sample on the far right is the MCH-only substrate. It is clear from the image that for samples 1 throught 8, the hybridization increases with increasing coverage.
For example number 8, the hybridization efficiency approaches 100%.
The amount of Hybridization for the samples with higher HS-ssDNA surface coverages is most probably due to steric and/or electrostatic interferences arising from the more tightly packed DNA monolayer. It is important to note that little if any radiolabled complement adsorbs on the MCH-only monolayer.
Electrochemistry was also used to quantitate hybridization as a function of HS-ssDNA surface density. Two different thiolated single-stranded DNA 25-mers were examined.
The sequence represented by the closed circles is the same as that used in all of the above-described experiments. The second sequence, represented by open circles, is a thiolated T25 homooligomer.
In this figure, the hybridized target (complement) density is plotted versus the surface immobilized probe density.
The dashed line in the figure represents the case where all of the surface-bound species are hybirdized with complement. From the firgure, it is easy to see that nearly 100% of the surface-bound DNA molecules are hybridized for coverage values less than 4 x 1012 molecules/cm2.
At coverages higher than this value, the hybridization efficiency to the differencies in binding specificity of the two sequences. That is, the hybridization conditions for the HS-T25 oligomer are not as rigid as that of the HS-ssDNA sequence.
Adam B. Steel, Tonya M. Herne, and Michael J. Tarlov, "Electrochemical Characterization and Quantitation of DNA Probes Immobilized on Gold Surfaces," submitted to Analytical Chemistry, 1998.
Tonya M. Herne and Michael J. Tarlov, "Characterization of DNA Probes Immobilized on Gold Surfaces," Journal of the American Chemical Society. 119, 8916-8920 (1997).
Kevin A. Peterlinz, Rosina M. Georgiadis, Tonya M. Herne, and Michael J. Tarlov, "Observation of Hybridization and Dehybridization of Thiol-tethered DNA Using Two Color Surface Plasmon Resonance Spectroscopy," Journal of the American Chemical Society, 119, 3401-3402 (1997).