Increasing the sensitivity of DNA microarrays by metal-enhanced fluorescence using surface-bound silver nanoparticles.
The effects of metal-enhanced fluorescence (MEF) have been measured for two dyes commonly used in DNA microarrays, Cy3 and Cy5. Silver island films (SIFs) grown on glass microscope slides were used as substrates for MEF DNA arrays. We examined MEF by spotting biotinylated, singly-labeled 23 bp DNAs onto avidin-coated SIF substrates. The fluorescence enhancement was found to be dependent on the DNA spotting concentration: below approximately 12.5 muM, MEF increased linearly, and at higher concentrations MEF remained at a constant maximum of 28-fold for Cy5 and 4-fold for Cy3, compared to avidin-coated glass substrates. Hybridization of singly-labeled oligonucleotides to arrayed single-stranded probes showed lower maximal MEF factors of 10-fold for Cy5 and 2.5-fold for Cy3, because of the smaller amount of immobilized fluorophores as a result of reduced surface hybridization efficiencies. We discuss how MEF can be used to increase the sensitivity of DNA arrays, especially for far red emitting fluorophores like Cy5, without significantly altering current microarray protocols.
Sabanayagam
Chandran R
CR
Department of Biochemistry and Molecular Biology, Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA. chandran@cfs.umbi.umd.edu
Lakowicz
Joseph R
JR
D002232:Carbocyanines / Q000737:chemistry* / Q000528:radiation effects; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000528:radiation effects; D005898:Glass / Q000737:chemistry; D053758:Nanoparticles / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D038761:Photobleaching; D012834:Silver / Q000737:chemistry*
D002232:Carbocyanines; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; C089187:cyanine dye 3; C085321:cyanine dye 5; D012834:Silver