Increasing the sensitivity of DNA microarrays by metal-enhanced fluorescence using surface-bound silver nanoparticles

Abstract

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.

Figure 1

Physical and optical properties of the SIF grown on glass substrates. (A) The top image is a field emission scanning electron micrograph of the SIF showing the heterogeneity of the particles' shapes and sizes. The inset is a higher magnification of the SIF. (B) Normalized absorption spectra (dotted lines) and fluorescence spectra (solid lines) for Cy3 (green) and Cy5 (red) labeled double-stranded DNAs in solution. Cy3 was excited with 520 nm, and Cy5 was excited with 610 nm. (C) Visible extinction spectra for the SIF substrate at different steps during functionalization: SIF (circles); SIF-biotin (dashed line) and SIF-biotin-avidin (squares). Extinction spectra were taken with 1 nm resolution. The experimental errors were similar for the three spectra, but error bars are shown for only the case of bare SIF, for graphical clarity.

Figure 2

Scheme showing DNA hybridization in solution versus on a surface. (A) Solution hybridization. Biotinylated probe oligonucleotides ‘b’ and fluorophore-labeled target oligonucleotides ‘F’ are mixed in an equimolar ratio, heated to 85°C, then slowly cooled to form fluorescently-labeled double-stranded DNA. After cooling to room temperature, the DNAs are arrayed onto avidin-coated microscope slides. (B) Surface hybridization. Biotinylated probe oligonucleotides are arrayed onto avidin-coated microscope slides. Fluorophore-labeled target oligonucleotides are deposited onto the array surface for hybridization at 45°C.

Figure 3

Solution hybridization. Intensity scans of Cy5- and Cy3-labeled double-stranded DNA arrayed onto SIF and glass substrates. Each data point represents an average of seven spots. Note the log scale on the x-axis. (A) Cy5 fluorescence on glass (filled circles) and SIF (empty circles). The solid lines are fits to Langmuir isotherms with k = 0.092 μM⁻¹. (B) Cy3 fluorescence on glass (filled circles) and SIF (empty circles). Fits to Langmuir isotherms yielded similar k values as obtained with Cy5. (C) Plots of the intensity enhancement factor versus spotting concentration. Cy5 is shown in red, and Cy3 is shown in green. The enhancement factor was determined by dividing the average spot intensity of SIF with the corresponding glass values.

Figure 4

Photostability of Cy5- and Cy3-labeled duplex DNA arrayed onto SIFs and glass substrates. (A) Cy3 fluorescence on glass (circles) and SIF (squares). (B) Cy5 fluorescence on glass (circles) and SIF (squares). Solid lines are exponential fits to the data (see text). (C) Plots of the cumulative intensities of Cy3 versus the number of scans on glass (circles) and SIF (squares). (D) Plots of the cumulative intensities of Cy5 versus the number of scans on glass (circles) and SIF (squares). Symbols are shown for every 10th data point for graphical clarity.

Figure 5

Fluorescence image of labeled oligonucleotide targets hybridized to MEF and glass DNA arrays. Probe oligonucleotides (23mer) were arrayed onto the substrates at different spotting concentrations: each row represents seven replicate spots at a given concentration; the rows are 2-fold serial dilutions starting with 50 μM (upper row). The image shows the Cy5 fluorescence as a result of co-hybridization with complementary Cy5- and Cy3-labeled targets (23mer). The scans were down sampled 5 × 5 pixels to plot the images in the figure, but for quantitation the original scan resolution (10 μm) was used (see Materials and Methods). The intensity bar shown in the lower right is a linear scale from 0 counts (black) to 34 000 counts (white).

Figure 6

Surface hybridization. Intensity scans of Cy5- and Cy3-labeled target oligonucleotides hybridized to probes arrayed onto SIF and glass substrates. Note the log scale on the x-axis. (A and B) show the average intensity versus spotting concentration for Cy5 and Cy3, respectively, on SIF substrates (empty circles) and glass (filled circles). (C) Plots of the intensity enhancement factor versus spotting concentration from the hybridization data. Cy5 is shown in red, and Cy3 is shown in green.