| Abstract
| - Performance improvements in DNA-modified surfaces required for microarray and biosensorapplications rely on improved capabilities to accurately characterize the chemistry and structure ofimmobilized DNA molecules on micropatterned surfaces. Recent innovations in imaging X-ray photoelectronspectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) now permit moredetailed studies of micropatterned surfaces. We have exploited the complementary information providedby imaging XPS and imaging TOF-SIMS to detail the chemical composition, spatial distribution, andhybridization efficiency of amine-terminated single-stranded DNA (ssDNA) bound to commercial polyacrylamide-based, amine-reactive microarray slides, immobilized in both macrospot and microarray diagnosticformats. Combinations of XPS imaging and small spot analysis were used to identify micropatterned DNAspots within printed DNA arrays on slide surfaces and quantify DNA elements within individual microarrayspots for determination of probe immobilization and hybridization efficiencies. This represents the first reportof imaging XPS of DNA immobilization and hybridization efficiencies for arrays fabricated on commercialmicroarray slides. Imaging TOF-SIMS provided distinct analytical data on the lateral distribution of DNAwithin single array microspots before and after target hybridization. Principal component analysis (PCA)applied to TOF-SIMS imaging datasets demonstrated that the combination of these two techniques providesinformation not readily observable in TOF-SIMS images alone, particularly in identifying species associatedwith array spot nonuniformities (e.g., “halo” or “donut” effects often observed in fluorescence images).Chemically specific spot images were compared to conventional fluorescence scanned images in microarraysto provide new information on spot-to-spot DNA variations that affect current diagnostic reliability, assayvariance, and sensitivity.
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