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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.

SUBMITTER: Lee CY

PROVIDER: S-EPMC2533279 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Publications

Fluorescence, XPS, and TOF-SIMS surface chemical state image analysis of DNA microarrays.

Lee Chi-Ying CY Harbers Gregory M GM Grainger David W DW Gamble Lara J LJ Castner David G DG

Journal of the American Chemical Society 20070711 30

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 imagi ...[more]

PMID: 17625851

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Project description:For immobilization of proteins onto surfaces in a specific and controlled manner, it is important to start with a well-defined surface that contains specific binding sites surrounded by a nonfouling background. For immobilizing histidine-tagged (his-tagged) proteins, surfaces containing nitrilotriacetic acid (NTA) headgroups and oligo(ethylene glycol) (OEG) moieties are a widely used model system. The surface composition, structure, and reactivity of mixed NTA/OEG self-assembled monolayers (SAMs) on Au substrates were characterized in detail using X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), time-of-flight secondary ion mass spectrometry (TOF-SIMS), and surface plasmon resonance (SPR) biosensoring. XPS results for sequential adsorption of NTA thiols followed by OEG thiols showed that OEG molecules were incorporated into an incompletely formed NTA monolayer until a complete mixed SAM was formed. The surface concentration of NTA headgroups was estimated to be 0.9-1.3 molecule/nm2 in the mixed NTA/OEG monolayers, compared to 1.9 molecule/nm2 in pure NTA monolayers. Angle-dependent XPS indicated NTA headgroups were slightly reoriented toward an upright position after OEG incorporation, and polarization-dependent NEXAFS results indicated increased ordering of the alkane chains of the molecules. Nitrogen-containing and OEG-related secondary ion fragments from the TOF-SIMS experiments confirmed the presence of NTA headgroups and OEG moieties in the monolayers. A multivariate peak intensity ratio was developed for estimating the relative NTA concentration in the outermost (10 A) of the monolayers. SPR measurements of a his-tagged, humanized anti-lysozyme variable fragment (HuLys Fv) immobilized onto Ni(II)-treated mixed NTA/OEG and pure NTA monolayers demonstrated the reversible, site-specific immobilization of his-tagged HuLys Fv (108-205 ng/cm2) with dissociation rates (koff) between 1.0 x 10-4 and 2.1 x 10-5 s-1, both depending on the NTA surface concentration and orientation. The monolayers without Ni(II) treatment exhibited low nonspecific adsorption of his-tagged HuLys Fv (<2 ng/cm2).

Dataset Information

Fluorescence, XPS, and TOF-SIMS surface chemical state image analysis of DNA microarrays.

Publications

Fluorescence, XPS, and TOF-SIMS surface chemical state image analysis of DNA microarrays.

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