Optimal Hybridization Efficiency Upon Immobilization of Oligonucleotide Double Helices

Abstract

For the first time, the covalent immobilization of oligonucleotides double helices onto surfaces prior to sequential denaturation and rehybridization is proven to lead to optimal hybridization efficiency. Two indirect methods were used for monitoring these reactions in situ: the quartz crystal microbalance with dissipation monitoring (QCM-D) and the wavelength interrogated optical sensor (WIOS, Bright Reader). Both techniques led to the result that with this immobilization approach one could reach nearly 100% hybridization efficiency. Moreover, applying the polymer theory to the adsorption of nucleotide sequences onto surfaces, we demonstrate, that for single stranded sequences the coil conformation prevails over the stretch one.

Bioinspired Quercitrin Nanocoatings: A Fluorescence-Based Method for Their Surface Quantification, and Their Effect on Stem Cell Adhesion and Differentiation to the Osteoblastic Lineage

“…This way, we expected that the use of the reducing agent NaCNBH 3 in the flavonoid grafting reaction would reduce the imine bond between quercitrin and the silane to a simple, less reactive, and irreversible C−N bond (QRred surfaces). Initially, we defined the concentration of reducing agent NaCNBH 3 as 50 μM, according to the work of Razumovitch et al, 29 and performed the quercitrin grafting reaction, followed by the addition of the reducing agent, in a two-step procedure. To evaluate the viability of this approach, we characterized the surfaces obtained by FTIR and XPS spectroscopy (Figure 2 and Figure 3).…”

A survey of the 2006–2009 quartz crystal microbalance biosensor literature

“…A number of publications describe the improvement of existing methods or development of new methods for the immobilization of RNA or DNA. These consisted of covalent immobilization (Lee et al ., 2008a; Razumovitch et al , 2009; Sato et al , 2009) including a photochemical method (Nakano et al , 2006), polycationic surfaces (Rawle et al , 2007; Yang et al , 2008a), inclusion in a layer‐by‐layer self‐assembly system (Sultan et al , 2009), natural organic matter (Nguyen and Chen, 2007; Nguyen and Elimelech, 2007a), adsorption directly to quartz (Nguyen and Elimelech, 2007b) and the use of biotinylated DNA on streptavidin (Yamasaki et al , 2007). Surface morphology was shown to play an important role, leading to a three‐ to five‐fold better sensitivity in an DNA detection assay where nanogold ‘hollow’ spheres where used instead of a flat gold surface (Lu et al ., 2007).…”

Bio-Decorated Polymer Membranes: A New Approach in Diagnostics and Therapeutics

“…Other types of biomolecules, such as antibodies [27] or DNA [28], were also successfully immobilized on a polymer membrane with the aim of developing highly active surfaces. A rabbit anti-goat antibody was successfully covalently immobilized on a nylon-grafted-glycidyl methacrylate polymer.…”

“…An aminopropyltritoxysilane-glutaraldehye surface functionalized with single-stranded DNA was able to recognize the complementary strand, which allowed for the development of a highly specific active surface with possible applications in medical domains [28]. Single-stranded nucleotides retain their coil conformation, whereas in the double-stranded form, the helical conformation is preferred.…”