ABSTRACT
Photochemical attachment of synthetic oligonucleotides on the three dimensional surface of a polyacrylamide based hydrogel was used in the specific detection of target oligonucleotides. Covalent attachment of the oligonucleotide to the hydrogel was mediated by the incorporation of a 2 + 2 photo-attachable functional group in both the hydrogel and the oligonucleotide probe. Expression and SNP assays were used to evaluate this platform.
Subject(s)
Hydrogels/chemistry , Oligonucleotide Array Sequence Analysis , Oligonucleotides/chemistry , Acrylic Resins/chemistry , Humans , Oligonucleotide Probes/chemistry , PhotochemistryABSTRACT
Using a fluorescence-based method, we have determined the number of thiol-derivatized single-stranded oligonucleotides bound to gold nanoparticles and their extent of hybridization with complementary oligonucleotides in solution. Oligonucleotide surface coverages of hexanethiol 12-mer oligonucleotides on gold nanoparticles (34 +/- 1 pmol/cm2) were significantly higher than on planar gold thin films (18 +/- 3 pmol/cm2), while the percentage of hybridizable strands on the gold nanoparticles (1.3 +/- 0.3 pmol/cm2, 4%) was lower than for gold thin films (6 +/- 2 pmol/cm2, 33%). A gradual increase in electrolyte concentration over the course of oligonucleotide deposition significantly increases surface coverage and consequently particle stability. In addition, oligonucleotide spacer sequences improve the hybridization efficiency of oligonucleotide-modified nanoparticles from approximately 4 to 44%. The surface coverage of recognition strands can be tailored using coadsorbed diluent oligonucleotides. This provides a means of indirectly controlling the average number of hybridized strands per nanoparticle. The work presented here has important implications with regard to understanding interactions between modified oligonucleotides and metal nanoparticles, as well as optimizing the sensitivity of gold nanoparticle-based oligonucleotide detection methods.
Subject(s)
Gold/chemistry , Nucleic Acid Hybridization , Oligonucleotides/chemistry , Spectrometry, Fluorescence/methods , Particle Size , Surface PropertiesABSTRACT
A new anchoring group is described for binding oligonucleotides to gold surfaces. On the basis of a ketal derived from 4,5-dihydroxy-1, 2 dithiane and epiandrosterone, it is easy to prepare and to link to oligonucleotides. Gold nanoparticle-oligonucleotide conjugates made using this cyclic disulfide linker serve as effective probes for detecting specific oligonucleotide sequences, and they exhibit much greater stability toward dithiothreitol than corresponding conjugates prepared with the conventional mercaptohexyl group or an acyclic disulfide unit. The high stability toward thiol deactivation likely results, in part at least, from anchoring each oligonucleotide to gold through two sulfur atoms.
Subject(s)
Disulfides/chemistry , Gold/chemistry , Oligonucleotides/chemistry , Steroids/chemistry , DNA Probes/chemical synthesis , Dithiothreitol/metabolism , Molecular Structure , Nucleic Acid HybridizationABSTRACT
A highly selective, colorimetric polynucleotide detection method based on mercaptoalkyloligonucleotide-modified gold nanoparticle probes is reported. Introduction of a single-stranded target oligonucleotide (30 bases) into a solution containing the appropriate probes resulted in the formation of a polymeric network of nanoparticles with a concomitant red-to-pinkish/purple color change. Hybridization was facilitated by freezing and thawing of the solutions, and the denaturation of these hybrid materials showed transition temperatures over a narrow range that allowed differentiation of a variety of imperfect targets. Transfer of the hybridization mixture to a reverse-phase silica plate resulted in a blue color upon drying that could be detected visually. The unoptimized system can detect about 10 femtomoles of an oligonucleotide.
