Attomole Detection of Mesophilic DNA Polymerase Products by Nanoparticle-Enhanced Surface Plasmon Resonance Imaging on Glassified Gold Surfaces

DNA microarrays are invaluable tools for biosensing applications such as diagnostic detection of DNA and analysis of gene expression. Surface plasmon resonance imaging can detect unlabeled oligonucleotide targets adsorbed to the array elements. The variety of biosensing applications can be expanded by enzymatic manipulation of DNA microarray elements, and the sensitivity of detection can be enhanced with the use of oligonucleotide immobilized onto a gold nanoparticle surface. We describe a novel method that couples a template-directed polymerase extension of a surface array element with nanoparticle-enhanced detection of the reaction product. Using this technique, it is possible to see as little as 10-100 amol of polymerase product, representing as little as 0.25% of a monolayer. This sensitivity would allow for the detection of a specific DNA target that is present in low amounts in a sample and with partially unknown sequence. One application of this method would be to identify the presence of the aberrantly recombined DNA sequences, such as those found in the fragile sites of chromosomes.