Fluoresceins, rhodamines, cyanines, and other similar fluorophores are widely used in biotechnology. The favorable aspects of these probes include large excitation coefficients and high quantum yields. An unfavorable property of these probes is the small Stokes’ shift and thus overlap of the absorption and emission spectra. As a result of the small Stokes’ shift, these probes display homo resonance energy transfer (RET), which results in self-quenching. In fact, self-quenching of fluorescein (Fl) is among the earliest observations in fluorescence spectroscopy.1–5
For fluorescein, the Forster distance for homo RET is ~42 Å,6
which is comparable to the size of DNA oligomers with ~13 base pairs.
At the present time, fluorescence is the primary detection method for genetic analysis, DNA sequencing, and DNA arrays. These arrays are widely used for high-throughput studies of gene expression7–9
and increasingly for medical testing, genetics profiling, or diagnostics.10,11
In all these uses it is desirable to obtain the largest possible signal per target strand. One obvious approach for larger signals is to label the DNA with more fluorophores. Unfortunately, this approach often results in self-quenching and decreased intensities.
In the present report, we describe a new approach to avoid self-quenching and to increase the brightness of highly labeled oligomers. We examined DNA 23-mers labeled with one or five fluorescein residues. These labeled oligomers were hybridized with a complementary biotinylated oligomer, which in turn was bound to a quartz substrate coated with biotinylated albumin and avidin. The double-stranded oligomers were examined on quartz and on quartz coated with metallic silver particles. The subwavelength- size silver particles were deposited by chemical reduction of silver. These particles are referred to as silver island films (SIFs) and are commonly used in surface-enhanced Raman scattering.12–14
In recent studies, we found that SIFs could result in increased intensities and increased photostability of nearby fluorophores.15,16
In the present report, we found that the emission intensities were increased 7- and 19-fold for the oligomers labeled with one or five fluoresceins, respectively. Photostability was also increased near the silver particles. These results suggest the use of silver particles or silver colloids on DNA array substrates for increased sensitivity.