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ABSTRACT: The ability of Bin+ cluster ion sources to provide information about the physical structure of an integrated film composed of oligonucleotides and non-nucleic acid oligomers of poly(2-hydroxyethyl methacrylate) (PHEMA) has been explored using static time-of-flight (ToF) SIMS (ToF-SIMS). Fragment intensities from PHEMA and oligonucleotides were compared. Negative ion spectra indicated that the Bi5+ source provided details that were either not apparent or at much reduced sensitivity when using Bi3+ and Bi+ sources. The Bi3+ ion source provided higher secondary ion intensities for positive ion fragments, and more than 200-fold selectivity for ionization of oligonucleotide in comparison to PHEMA when in mixture, even though the oligonucleotides were not immobilized as a coating superimposed on PHEMA. Copyright © 2010 John Wiley & Sons, Ltd.
Surface and Interface Analysis 05/2010; 43(1‐2):322 - 325. · 1.18 Impact Factor
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ABSTRACT: Preliminary studies of mixed films composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) (PHEMA) have recently been shown to enhance the selectivity for detection of 3 base-pair mismatched (3 bpm) oligonucleotide targets. Evaluation of selectivity for detection of single nucleotide polymorphisms (SNP) using such mixed films has now been completed. The selectivity was quantitatively determined by considering the sharpness of melt curves and melting temperature differences (DeltaT(m)) for fully complementary targets and SNPs. Stringency conditions were investigated, and it was determined that the selectivity was maximized when a moderate ionic strength was used (0.1-0.6 M). Increases of DeltaT(m) when using mixed films were up to 3-fold larger compared to surfaces containing only immobilized oligonucleotide probes. Concurrently, increases in sharpness of melt curves for 1 bpm targets were observed to be up to 2-fold greater for mixed films. The co-immobilization of PHEMA resulted in a more homogeneous distribution of oligonucleotide probes on surfaces. Lifetime measurements of fluorescence emission from immobilized oligonucleotide probes labeled with Cy3 dye indicated the difference in microenvironment of immobilized oligonucleotides in the presence of PHEMA.
Analytica chimica acta 02/2010; 661(1):103-10. · 4.31 Impact Factor
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ABSTRACT: Covalently immobilized mixed films of oligonucleotide and oligomer components on glass and silicon surfaces are reported. This work has investigated how such films can improve selectivity for the detection of multiple base-pair mismatches. The intention was to introduce a "matrix isolation" effect on oligonucleotide probe molecules by surrounding the probes with oligomers, thereby reducing oligonucleotide-to-oligonucleotide and/or oligonucleotide-to-surface interactions. Thiol-functionalized oligonucleotide was coupled onto (3-aminopropyl)trimethoxysilane (APTMS) via a heterobifunctional linker, succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC). Using a variety of monomers such as 2-hydroxyethyl methacrylate (HEMA), oligomers were grown by surface-initiated atom transfer radical polymerization (ATRP) from a bromoisobutyryl NHS ester initiator which was immobilized onto APTMS sites that coated glass and oxidized silicon substrates. Various surface modification steps on silicon substrates were characterized by ellipsometry, wettability, atomic force microscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry. Polymerized HEMA (PHEMA) in mixture with oligonucleotide probes was evaluated for fluorescence transduction of hybridization. The presence of PHEMA was found to provide a sharper melt curve for hybrids containing both fully complementary and three base-pair mismatched targets, and this surface derivatization also minimized non-selective adsorption. The maximum increase in slope was improvement by a factor of 3-fold. An increase of up to 30% in difference of melting temperatures between fully complementary and 3 base-pair mismatched targets was achieved using PHEMA. The results suggest that the presence of oligomers dispersed among DNA hybrids can improve selectivity through what is believed to be a reduction of dispersity of interactions of probes with targets, and probes within their local environment at a surface.
Analytica chimica acta 05/2009; 639(1-2):1-12. · 4.31 Impact Factor
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ABSTRACT: Silane coupling agents are commonly used to activate surfaces for subsequent immobilization of biomolecules. The homogeneity and surface morphology of silane films is important for controlling the structural order of immobilized single-stranded DNA probes based on oligonucleotides. The surfaces of silicon wafers and glass slides with covalently attached 3-glycidoxypropyltrimethoxysilane (GOPS) have been characterized by using angularly dependent X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (ToF-SIMS), atomic force microscopy (AFM), scanning electron microscopy (SEM), and monochromatic and spectroscopic ellipsometry. XPS and ToF-SIMS data provided evidence of complete surface coverage by GOPS. Data from angularly resolved XPS and ellipsometry methods suggested that the GOPS films were of monolayer thickness. AFM and SEM data indicated the presence of films that consisted of nodules approximately 50-100 nm in diameter. Modeling suggested that the nodules may lead to a nanoscale structural morphology that might influence the hybridization kinetics and thermodynamics of immobilized oligonucleotides.
Analytical and Bioanalytical Chemistry 10/2005; 383(2):187-200. · 3.78 Impact Factor
