By engineering PC resonances for compatibility with a commercial laser scanner, the benefits of enhanced fluorescence can be applied to a standard microarray experiment with no changes to the experimental protocol. While the initial photolithography process needed to fabricate the silicon mold of the grating has high costs, a single round of photolithography on silicon can be translated into thousands of devices that are fabricated uniformly over large areas. By fabricating the mold on an 8 in. wafer, there is a large degree of flexibility in fitting PCs to preferred labware formats such as microscope slides and microtiter plates. Because PCs were cut to fit standard microscope slides, they could be processed with existing protocols and scanned with commercially available equipment, allowing for convenient adoption of PC substrates in a standardized experiment. Not only does the nanoreplica molding process provide a convenient form factor for the substrates, it also enables the excellent level of optical uniformity required to ensure that every spot on the microarray experiences the same level of enhancement. This is key to ensure that the data obtained from PCs does not have a higher level of variation than the data obtained from glass slides.
The signal enhancement factor primarily used in previous work in this field is defined as spot intensity subtracted by the local background observed on the enhancement substrate divided by the same value observed on the glass slide or control substrate. The signal enhancement factor observed from Cy-5 spots with high expression genes in this microarray experiment was approximately 60×, which is identical to the enhancement demonstrated in previous work with this substrate.14
Thus, the PC signal enhancement compares favorably to experiments with metal island films that have yielded signal enhancement factors of 10–40×.5,6
However, the signal enhancement factor is not an ideal measurement to assess the practical utility of the substrate. This work has focused on SNR enhancement rather than signal enhancement because microarray data analysis programs use SNR values to classify spots as detected or not detected. It is possible to achieve good signal enhancement without achieving similar SNR enhancement if a substrate enhances fluorescence but has a large noise value thus voiding any advantages of fluorescence enhancement. Without knowledge of a substrate's impact on the SNR observed from spots, it is difficult to ascertain whether a substrate will benefit a target assay. The PC not only attains a large signal enhancement but it also achieves an SNR enhancement of approximately 10× (measured over all spots in the experiment), suggesting that the array can detect hybridization at concentrations 10× lower than can be detected on glass substrates.
The noise in a DNA microarray experiment arises primarily from the following sources: sample variation, nonspecific binding, instrumentation, and substrate fluorescence. Variation in the amount of nucleic acid sample captured is accounted for by hybridizing multiple arrays and figures prominently into tests of significance for differential expression experiments. Nonspecific binding is controlled largely by blocking and hybridization conditions and is assessed by evaluating negative control spots. The noise observed from instrumentation can be characterized by measurements of dark noise, but in this experiment, this represents only <5 counts relative to substrate noise levels more than 5× greater than this value. Substrate fluorescence is typically not manipulated because most microarray protocols have been optimized for a few common substrates. For genes with low expression levels, however, substrate fluorescence can be a significant contributor to noise. Changes in expression may not be large enough to overcome the noise in normal substrates, despite the fact these genes may be just as important as high expressors for cellular function.
Utilizing PCs as substrates amplifies the fluorophore intensity relative to the substrate fluorescence intensity and decreases the impact of substrate fluorescence on the measurements. Because the signal-to-noise ratio is enhanced by more than 1 order of magnitude on PC substrates, genes with expression levels that were lower than the noise floor on glass substrates can now be measured on PCs. This allows researchers to preserve the advantageous throughput of microarrays while increasing the sensitivity of their measurements. The practical effect of the PC is to improve the dynamic range of the expression measurements and allow for quantification of low expression genes. These low expression genes are not only detected, as evidenced by the increased number of genes above the SNR threshold, but also changes in the expression of these genes can be observed in the context of statistical testing. The direction of differential expression in these low expression genes is confirmed by sequencing data, which agreed with the microarray analysis for 39 of the 41 genes identified as differentially expressed only on PC microarrays. The capability of the PCs to measure low expression genes is reflected by the differences in average expression intensity between genes that were classified as differentially expressed only on the PCs (Table S-2 in the Supporting Information
), with an average intensity of 148 counts for the seed sample genes and an average intensity of 212 counts for the leaf sample genes, compared to genes classified as differentially expressed on glass slides (Table S-1 in the Supporting Information
), with an average intensity of 2930 counts for the seed sample genes and 2830 counts for the leaf sample genes. This finding is validated by the sequencing data as well. The 41 genes in Tables S-4 and S-2 in the Supporting Information
corresponding to genes detected as differentially expressed on the PC slides had an average RPKM value of 56 in the leaf sample and 67 in the seed sample, whereas the 26 genes detected as differentially expressed on both PC and glass slides had much higher average RPKMs in both the leaf (341 PRKM) and seed (3965 RPKM) samples, respectively. Thus, both microarray and sequencing data suggest the PC can reliably quantify genes with expression levels at least 1 order of magnitude lower than measured with conventional glass microarrays. By expanding the dynamic range of the microarray experiment, the number of genes for which statistically significant changes in expression could be observed improved from 27 to 68 genes, or from 13 to 34% of the genes probed in the experiment. Because the gene expression follows a power law distribution, modest enhancements in the performance of the assay can dramatically increase the number of genes researchers are able to probe in this microarray format. This data thus suggests that the detection capabilities of microarray protocols currently used today can be greatly expanded by substitution of conventional substrates with enhanced fluorescence substrates such as PCs.
The increased SNRs provided by PCs may allow researchers to perform experiments that are currently problematic on glass slides. Because lower amounts of bound sample can be detected with the PC, sample sizes may be reduced to volumes that would be difficult to probe using normal glass substrates. This may be particularly helpful for profiling gene expression in smaller tissue samples or small populations of rare cells such as stem cells. Alternately, the reduction in experimental variation afforded by this substrate may allow researchers to confidently identify differentially expressed genes with fewer replicates, which may also prove useful with small sample sizes or rare cells. This approach is not limited to conventional DNA microarray experiments. Any surface-bound biomolecular assay can be performed on these PCs for improved performance, as is illustrated in previous work with immunoassays.25
This substrate can also potentially be adapted to improve reliability of novel technologies such as next-generation genomic sequencing platforms, since these instruments make extensive use of fluorescent molecules.