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

Abstract

DNA microarrays are invaluable tools for biosensing applications such as diagnostic detection of DNA and analysis of gene expression. Surface plasmon resonance imaging (SPRI) 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 (detector-NP). 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 attomoles 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.

Two-dimensional enzyme diffusion in laterally confined DNA monolayers

“…The side-access effect reported in this study has not been hitherto described, although other investigations demonstrated enzymatic reactions on surface-bound DNA molecules 6,[21][22][23][38][39][40][41][42][43][44] . These studies used either longer dsDNA molecules 23,40 or molecules provided with significantly less-rigid surface linkers and/ or different chemical strategies for DNA self-assembly 6,23,39,41 .…”

“…These studies used either longer dsDNA molecules 23,40 or molecules provided with significantly less-rigid surface linkers and/ or different chemical strategies for DNA self-assembly 6,23,39,41 . Several studies involved ligation enzymes 42,43 , the reactions of which occurred on DNA-binding sites that are readily available on the topmost interface of the matrices.…”

“…In biotechnology of synthetic systems, a current trend is to exploit the possibility of assembling functional biomolecules within prototypical devices in which the levels of crowding and compartmentalization are representative of those of living systems [3][4][5][6][7][8][9][10][11][12][13][14][15] . Little is known about the effect of confinement on the properties and functions of these molecules within such crowded systems 1,2 ; thus, novel in vitro approaches can provide key information in aiding future biotechnology developments.…”

“…The immobilization of dense DNA structures on solid supports, obtained by self-assembly and enzymatic manipulations, holds a central role as a tool for developing miniaturized devices with innovative applications, ranging from biosensing [6][7][8][9] to next-generation information technology 14,15 . For example, restriction enzymes that cleave double-stranded (ds)DNA molecules are widely utilized in cloning as well as in a number of DNA-based assays [16][17][18] because they target very specific dsDNA sites.…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…An advantage of the SPR-assisted detection system is the ability to detect amplification products in nontransparent media which, for example, could be potentially useful in monitoring direct PCR amplification from blood [93,94]. A similar SPR detection technique has been utilized for isothermal amplification kinetic monitoring [74,95,96]. …”