Hybridization Behavior of Mixed DNA/Alkylthiol Monolayers on Gold:  Characterization by Surface Plasmon Resonance and ³²P Radiometric Assay

Abstract

Nucleic acid assay from a complex biological milieu is attractive but currently difficult and far from routine. In this study, DNA hybridization from serum dilutions into mixed DNA/mercaptoundecanol (MCU) adlayers on gold was monitored by surface plasmon resonance (SPR). Immobilized DNA probe and hybridized target densities on these surfaces were quantified using 32P-radiometric assays as a function of MCU diluent exposure. SPR surface capture results correlated with radiometric analysis for hybridization performance, demonstrating a maximum DNA hybridization on DNA/MCU mixed adlayers. The maximum target surface capture produced by MCU addition to the DNA probe layer correlates with structural and conformational data on identical mixed DNA/MCU adlayers on gold derived from XPS, NEXAFS, and fluorescence intensity measurements reported in a related study (Lee, C.-Y.; Gong, P.; Harbers, G. M.; Grainger, D. W.; Castner, D. G.; Gamble, L. J. Anal. Chem. 2006, 78, 3316-3325.). MCU addition into the DNA adlayer on gold also improved surface resistance to both nonspecific DNA and serum protein adsorption. Target DNA hybridization from serum dilutions was monitored with SPR on the optimally mixed DNA/MCU adlayers. Both hybridization kinetics and efficiency were strongly affected by nonspecific protein adsorption from a complex milieu even at a minimal serum concentration (e.g., 1%). No target hybridization was detected in SPR assays from serum concentrations above 30%, indicating nonspecific protein adsorption interference of DNA capture and hybridization from complex milieu. Removal of nonsignal proteins from nucleic acid targets prior to assay represents a significant issue for direct sample-to-assay nucleic acid diagnostics from food, blood, tissue, PCR mixtures, and many other biologically complex sample formats.

Effect of Molecular Crowding on the Response of an Electrochemical DNA Sensor

“…We find that the highest signal suppression is obtained with the highest probe density investigated, despite the fact that hybridization efficiency may be reduced as probe density increases. 5,[29][30][31][32][33][34][35][36] In contrast to the improved signal suppression observed at the highest probe densities, we find that sensor equilibration time slows monotonically with increasing density. Finally, we find that the specificity of hybridization is not significantly affected by changes in probe density.…”

“…5,[29][30][31][32][33][34][35][36] The probe density dependence of E-DNA signaling is more complex. For example, we find that the highest probe densities we investigated (~2.1 × 10 12 molecules/cm 2 ) produce the largest signal suppression (~71% at our test target concentration) ( Figure 3 and Figure 4, Table 4).…”

“…Nevertheless, key issues in their fabrication and use have not yet been explored in detail. For example, while it is likely that the signaling properties of these sensors depend sensitively on the density of immobilized probe DNA molecules on the sensor surface (measured in molecules of probe per square centimeter) [see, e.g., refs 5 and [29][30][31][32][33][34][35][36] ], no systematic study of this effect has been reported. Similarly, while it appears that the size of the target and the location of the recognition element within the target sequence affect signal suppression, 24 this effect, too, has seen relatively little study.…”

Scaffolded biosensors with designed DNA nanostructures

“…The use of MCH was later expanded with other diluent thiols. 31,32 An example of this was a mixed ssDNA/oligo-ethylene glycol monolayer that was used to improve the protein resistance of the surface, which makes it broadly applicable in biological medium. 33 The DNA probe density is a controlling factor for the efficiency of the target capture, as well as for the kinetics of the target/probe hybridization.…”

Construction of a densely poly(ethylene glycol)-chain-tethered surface and its performance

“…In a common method for the passivation of gold surfaces, ssDNA-SH-modified gold surfaces can be exposed to a solution containing a small-molecular-weight sulfanyl compound, such as mercapto hexanol or undecanol, which displaces most of the ssDNA-SH from the surface and forces the remaining ssDNA into an upright conformation. [66][67][68][69][70][71] Because this method is based on the substitution reaction between ssDNA-SH and sulfanyl-anchored small molecules, the decrease in the surface density of the ssDNA-SH is inevitable, resulting in low sensitivity for the constructed DNA-based sensor.…”