Structure and DNA Hybridization Properties of Mixed Nucleic Acid/Maleimide−Ethylene Glycol Monolayers

• Published in: Analytical chemistry (Washington) Vol. 79; no. 12; pp. 4390 - 4400
• Main Authors: Lee, Chi-Ying, Nguyen, Phuong-Cac T, Grainger, David W, Gamble, Lara J, Castner, David G
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.06.2007
• Subjects: ABSORPTION; ADSORPTION; Analytical chemistry; BASIC BIOLOGICAL SCIENCES; BLOOD SERUM; BUFFERS; CHEMISTRY; Deoxyribonucleic acid; DETECTION; DISULFIDES; DNA; DNA - analysis; DNA - chemistry; DNA HYBRIDIZATION; DNA Probes; DNA, Single-Stranded - chemistry; EFFICIENCY; Ethylene Glycol - chemistry; Exact sciences and technology; FINE STRUCTURE; GLYCOLS; GOLD; Gold - chemistry; HYBRIDIZATION; Maleimides - chemistry; Mass spectrometry; MASS SPECTROSCOPY; MIXTURES; MONITORING; national synchrotron light source; Nucleic Acid Hybridization; ORIENTATION; Osmolar Concentration; PHYSICS OF ELEMENTARY PARTICLES AND FIELDS; PLASMONS; POLARIZATION; PROBES; PROTEINS; RESONANCE; Spectrometric and optical methods; Spectrometry, Mass, Secondary Ion; Spectrum Analysis; Sulfhydryl Compounds - chemistry; Surface chemistry; Surface Properties; Temperature; Time Factors; X-RAY PHOTOELECTRON SPECTROSCOPY; X-Rays; Performance evaluation; Monolayer; Ethylene glycol; Gold; Chemical analysis; Immobilization; X ray spectrometry; Secondary ion mass spectrometry; Protein; Blood; Chemical enrichment; Surface structure; Biological compound; Adsorption; Efficiency; Ionic strength; Time of flight method; DNA; Serum; Surface plasmon resonance; DNA hybridization; Nucleic acid; Monitoring
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac0703395

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 and time-of-flight secondary ion mass spectrometry. 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 toward 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 amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.