Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy

TitleDetection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy
Publication TypeJournal Article
Year of Publication2007
AuthorsKang SH, Kim YJ, Yeung ES
Journal TitleAnalytical and Bioanalytical Chemistry
Volume387
Pages2663-2671
Date PublishedApr
Type of ArticleArticle
ISBN Number1618-2642
Accession NumberISI:000245123600010
KeywordsDIP-PEN NANOLITHOGRAPHY, dual color, DYNAM, GENE-EXPRESSION, hybridization, IMMOBILIZATION, interface, internal reflection fluorescence microscopy (TIRFM), LIVING CELLS, microarrays, nanoarray chip, NANOARRAYS, PLASMA-MEMBRANE, single DNA molecules, total
Abstract

We examined the use of prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) to probe DNA molecules at the single-molecule level. The system allowed the direct detection of the complementary interactions between single-stranded probe DNA molecules (16-mer) and various lengths of single-stranded target DNA molecules (16-mer and 55-mer) that had been labeled with different fluorescent dyes (Cy3, Cy5, and fluorescein). The polymer-modified glass substrate and the extent of DNA probe immobilization were easily characterized either with standard TIRFM or with atomic force microscopy. However, only dual-color TIRFM could provide unambiguous images of individual single-stranded target DNA molecules hybridized with the correct sequence in the range of fM-aM. Succinic anhydride showed low RMS roughness and was found to be an optimal blocking reagent against non-specific adsorption, with an efficiency of 92%. This study provides a benchmark for directly monitoring the interactions and the detection of co-localization of two different DNA molecules and can be applied to the development of a nanoarray biochip at the single-molecule level.

DOI10.1007/s00216-007-1134-1
Alternate JournalAnal. Bioanal. Chem.