Development, characterization, and optimization of a nanoparticle-based DNA hybridization detection strategy

Gold colloids were used as target labels in an attempt to improve the sensitivity and selectivity of the existing DNA microarray technology. Two synthetic routes, which could be used indistinctly from each other, were used to assemble these colloidal “arrays”. The sequential route calls for the successive deposition, onto a gold substrate, of (1) a ssDNA oligonucleotide, (2) a 6-mercapto-l-hexanol (MH) passivating layer, (3) the complementary ssDNA oligomer (target), and (4) the colloidal label. Alternatively, the co-deposition route calls for the immersion of a gold substrate in a mixture of (1) and (2), followed by hybridization of gold nanoparticles, each derivatized with a only a few ssDNA targets.; dsDNA-nanoparticle monolayers were systematically characterized, using STM, vibrational spectroscopies and chronocoulometry, in order to determine the factors affecting surface coverage, a parameter weighting on both sensitivity and selectivity. These factors included probe density, target concentration and colloid size. Surface coverage was found to increase linearly with probe density, while target hybridization showed a cooperative behavior as a function of concentration. It was also determined that smaller nanoparticles were suitable to form dense and homogeneous dsDNA-nanoparticle monolayers and to prevent the formation of large, non selective aggregates. We have shown that once bound to the substrate, nanoparticles were electrically isolated from their neighbors and that they enhanced the spectral signature of the hybrids or of the molecules they were coated with.; The hybridization efficiency of our technique was, because of the sheer size of the particle and of its ionic atmosphere, orders of etudes lower than what is typically reported for DNA microarrays. Despite this low hybridization efficiency, target hybridization could still be easily detected.; Finally, we found that ssDNA probes desorbed from the gold substrate, during hybridization, at temperatures as low as 45°C and that the salinity of the hybridization buffer had some effect on the rate of probe desorption.; This study has important implications with regard to designing more sensitive and selective DNA microarrays as it introduces a new approach to DNA hybridization detection and provides, for the first time, an exhaustive insight into the parameters affecting array performance.