Sensitive Detection of DNA Immobilization and Hybridization on Polyelectrolyte-Modified Surfaces

Accurate identification and quantification of nucleic acid targets has been a major issue in biological sciences, as such targets reflect, in many cases, the state of the biological system. Specific and predictable immobilization of oligonucleotides and their hybridization with respective counterparts play a fundamental role in design of new oligonucleotide microarrays for DNA biosensing. However, signal interpretation generated by a microarray has often been challenging in terms of the surface chemistry involved, as various details of the DNA organization on surface haven’t been well understood.

Immobilization of oligo-DNA on surfaces using the polyelectrolyte layer-by-layer (LbL) method provides a simple and convenient approach to DNA application in sensing schemes. Here we demonstrate DNA strands immobilization on glass substrates and on gold nano-island films prepared by evaporation on glass and annealing, using the polyelectrolyte LbL approach. The Au nano-island films serve as localized surface plasmon resonance (LSPR) transducers, enabling optical detection of DNA binding. Use of DNA strands labeled with chromophores allows quantification of DNA immobilization and surface hybridization with complimentary or mismatched strands. Hence, each surface modification step (polyelecrolyte adsorption, immobilization of labeled ssDNA probe, hybridization with target DNA) can be monitored by spectroscopic detection of the Au LSPR response and by the chromophore UV-Vis absorbance.

Quantification of the DNA immobilization on the surface and subsequent hybridization, performed as described above, was verified by fluorescence measurements, carried out using the fluorescent properties of the same chromophore-derivatized DNA strands.

We show that optical signal associated with DNA sensing is sensitive to various experimental conditions (polyelectrolyte organization, DNA immobilization conditions, additional compounds or counter ions), allowing the development of a strategy for optimized DNA detection on polyelectrolyte-modified surfaces.