Structure and DNA hybridization properties of mixed nucleic acid/maleimide-ethylene glycol monolayers
| Autor: | David G. Castner, Chi Ying Lee, Phuong Cac T Nguyen, David W. Grainger, Lara J. Gamble |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Ethylene Glycol
Time Factors Surface Properties Analytical chemistry DNA Single-Stranded Spectrometry Mass Secondary Ion Buffers Article Analytical Chemistry Maleimides Nucleic acid thermodynamics chemistry.chemical_compound Blood serum Monolayer Sulfhydryl Compounds Surface plasmon resonance Maleimide Hybridization probe Spectrum Analysis X-Rays Osmolar Concentration Temperature Nucleic Acid Hybridization DNA chemistry Biophysics Gold DNA Probes Ethylene glycol |
| Zdroj: | Analytical chemistry. 79(12) |
| ISSN: | 0003-2700 |
| Popis: | The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Orientation of the ssDNA probes was determined by near edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s → π* transition) indicate that the immobilized ssDNA molecules reorient towards a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the “high density” probe surface than on the “high efficiency” probe surface. The amount of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to non-specific serum protein adsorption onto the sensing surface. |
| Databáze: | OpenAIRE |
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