Improved sensitivity of DNA microarrays using photonic crystal enhanced fluorescence

Abstract

DNA microarrays are used to profile changes in gene expression between samples in a high-throughput manner, but measurements of genes with low expression levels can be problematic with standard microarray substrates. In this work, we expand the detection capabilities of a standard microarray experiment using a photonic crystal (PC) surface that enhances fluorescence observed from microarray spots. This PC is inexpensively and uniformly fabricated using a nanoreplica molding technique, with very little variation in its optical properties within- and between-devices. By using standard protocols to process glass microarray substrates in parallel with PCs, we evaluated the impact of this substrate on a one-color microarray experiment comparing gene expression in two developmental stages of Glycine max. The PCs enhanced the signal-to-noise ratio observed from microarray spots by 1 order of magnitude, significantly increasing the number of genes detected above substrate fluorescence noise. PC substrates more than double the number of genes classified as differentially expressed, detecting changes in expression even for low expression genes. This approach increases the dynamic range of a surface-bound fluorescence-based assay to reliably quantify small quantities of DNA that would be impossible with standard substrates.

Figure 1

(a) Schematic of PC design dimensions. (b) Atomic force micrograph of completed PC structure (after TiO₂ deposition), with a measured period of 366 nm and height of 50 nm.

Figure 2

Optical transmission measurements from all six PCs (each represented by a colored solid line) used in this study, obtained by illuminating the devices with polarized, collimated white light. Resonances with narrow spectral features are excited when the PCs are illuminated with transverse magnetic polarized light and overlap the excitation wavelength of 632.8 nm (dotted line). Resonances with broader spectral features are excited when the PCs are illuminated with transverse electric polarized light and overlap the emission filter wavelengths of 670–710 nm (dotted box).

Figure 3

Fluorescence images at identical gains of a single identical microarray grid on glass (a) and PC (b), with brightness and contrast adjustment to make the maximum number of spots visible on both images. For comparison of spot intensities, line profiles of identical locations on the grid for glass and PC are illustrated on the same plots. Lower expression genes appear in (c) and higher expression genes appear in (d).

Figure 4

Logarithmic plots of duplicate-averaged SNR values for the 192 probed genes on selected glass–PC slide pairs for each tissue. Genes are organized in decreasing expression order for each chip, and an SNR detection threshold of 3 appears as the cutoff line in each graph. SNR expression profiles for cotyledon RNA appear in (a) and (c) for a glass slide and its paired PC, respectively. Trifoliate RNA expression profiles are plotted in (b) and (d) for a glass slide and its paired PC, respectively. Negative control spots on all slides appeared below the detection threshold.

Figure 5

Volcano plots detailing the relationship between fold-change and inverse p-value to assess differential expression between the trifoliate and cotyledon samples, with positive fold changes indicating increased trifoliate expression and negative fold changes indicating increased cotyledon expression. Green vertical lines represent the 2-fold change cutoff, and the yellow horizontal line denotes a p-value cutoff of 0.05. Genes meeting both thresholds are indicated by red spots. Unaveraged data representing all 7680 spots across all experimental slides (3 replicates per tissue) appear in (a) for the glass slides and (b) for the PCs, with 865 spots differentially expressed on glass slides and 1431 spots on the PCs. Averaging within-slide repeats condensed the data to 192 distinct genes and controls, which appear in (c) for the glass slides and (d) for the PCs. Of the 192 genes probed, 27 were classified as differentially expressed on the glass slides, while 68 met this classification on the PCs.

Figure 6

Logarithmic plot of fold change comparisons between glass microarray, PC microarray, and sequencing results for five genes randomly selected from the list of genes found to be differentially expressed on the PCs but not on the glass slides. Fold change values for glass and PC microarrays were calculated by determining the ratio between average microarray expression level for trifoliate samples to average microarray expression level for cotyledon samples. A similar ratio was calculated using number of reads for sequencing data. The PC microarray results show similar directions and magnitudes of change compared to sequencing data for all 5 genes, while glass microarray data for CHP089 and CHP004 does not agree with the sequencing data.