Fluorescence, XPS, and TOF-SIMS Surface Chemical State Image Analysis of DNA Microarrays

Abstract

Performance improvements in DNA-modified surfaces required for microarray and biosensor applications rely on improved capabilities to accurately characterize the chemistry and structure of immobilized DNA molecules on micropatterned surfaces. Recent innovations in imaging X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) now permit more detailed studies of micropatterned surfaces. We have exploited the complementary information provided by imaging XPS and imaging TOF-SIMS to detail the chemical composition, spatial distribution, and hybridization efficiency of amine-terminated single-stranded DNA (ssDNA) bound to commercial polyacrylamide-based, amine-reactive microarray slides, immobilized in both macrospot and microarray diagnostic formats. Combinations of XPS imaging and small spot analysis were used to identify micropatterned DNA spots within printed DNA arrays on slide surfaces and quantify DNA elements within individual microarray spots for determination of probe immobilization and hybridization efficiencies. This represents the first report of imaging XPS of DNA immobilization and hybridization efficiencies for arrays fabricated on commercial microarray slides. Imaging TOF-SIMS provided distinct analytical data on the lateral distribution of DNA within 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 provides information not readily observable in TOF-SIMS images alone, particularly in identifying species associated with 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 microarrays to provide new information on spot-to-spot DNA variations that affect current diagnostic reliability, assay variance, and sensitivity.

Multi-technique Characterization of DNA-Modified Surfaces for Biosensing and Diagnostic Applications

“…Prior to exposing the microarray slide to complementary target capture, similar XPS P 2p, N 1s, and Na 1s signal intensities were detected from all microspots, regardless of whether the microspots were printed utilizing complementary or noncomplementary probe sequences. After target hybridization, microspots with complementary probe sequences had higher XPS P 2p, N 1s, and Na 1s signal intensities compared to microspots with noncomplementary probe sequences, as expected [ 71 ]. , and sodium (Na1s) with the substrate silicon (Si2p) signal intensity images (800 μm × 800 μm) from printed DNA probes on CodeLink™ polymer microarray slides before ( a ) and after ( b ) DNA target hybridization.…”

“…Previous studies have shown that static ToF-SIMS, in combination with multivariate analysis statistical methods such as principal component analysis (PCA), can provide the distribution of chemical species across a patterned surface at a submicrometer resolution [ 69 , 70 ]. An example of the value of the dual use of imaging XPS and imaging ToF-SIMS to determine the chemical composition, spatial distribution, and hybridization effi ciency of amine-terminated ssDNA bound to commercial polyacrylamide-based microarray slides was published in 2007 [ 71 ]. Figure 11.7 shows XPS elemental images (P, N, Na, and Si) of noncontact printed DNA microarray spots (100-150-μm diameter) on CodeLink™ (GE Amersham microarray substrate) slides [ 55 , 71 ].…”

“…% of probe spot) − 1] × 100 %} (Reproduced with permission from Ref. [ 71 ]. Copyright © 2007 American Chemical Society)" Small-area region-of-interest (ROI) analyses were performed to obtain quantitative elemental composition information about individual DNA spots both before and after hybridization.…”

“…The DNA surface coverage is proportional to the phosphorus atomic concentration, as has been shown previously for immobilized DNA on gold [ 25 , 61 ] and polymer-modifi ed silicon substrates [ 55 , 58 ]. Figure 11.8c shows relative amounts of surface-immobilized probe and hybridized DNA obtained for each probe printing concentration from small-area XPS analysis [ 71 ]. Figure 11.8c shows the P 2p at.% from the DNA microspots' increases with increasing spotting-probe solution concentration.…”

Adding Spatial Control to Click Chemistry: Phototriggered Diels–Alder Surface (Bio)functionalization at Ambient Temperature

“…Imaging timeof-flight secondary-ion mass spectrometry (ToF-SIMS) is a commonly utilized and sensitive technique that allows the highly spatially resolved analysis of molecular patterns on solid substrates. [13] In contrast to traditional fluorescence imaging, ToF-SIMS data provides detailed information on the chemical composition essential for analysis of non-fluorescent (bio)molecules. For instance, bromine compounds with their inherent isotopic pattern can be unambiguously detected by ToF-SIMS; therefore, the bromine-containing dienophile 7 ( Figure 2) was utilized in the current study as a molecular marker to spatially map the locally constrained surface grafting.…”