Surface Plasmon Resonance Imaging Measurements of DNA and RNA Hybridization Adsorption onto DNA Microarrays

TLDR

SPR imaging is a surface‑sensitive technique that measures interactions of unlabeled biomolecules with surface‑bound arrays. The study applies SPR imaging to quantitatively detect low‑concentration hybridization adsorption of short DNA and RNA oligonucleotides, including 16S rRNA, onto a DNA microarray. High‑density thiol‑DNA probes covalently bound to gold enable stable, repeatable hybridization, while Fresnel‑based reflectivity analysis yields a linear relationship that allows construction of a quantitative adsorption isotherm. Both DNA and RNA oligonucleotides were detected down to 10 nM (2 nM for 16S rRNA), and their adsorption followed a Langmuir isotherm with an affinity coefficient of 1.8 × 10⁷ M⁻¹.

Abstract

Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic technique for measuring interactions between unlabeled biological molecules with arrays of surface-bound species. In this paper, SPR imaging is used to quantitatively detect the hybridization adsorption of short (18-base) unlabeled DNA oligonucleotides at low concentration, as well as, for the first time, the hybridization adsorption of unlabeled RNA oligonucleotides and larger 16S ribosomal RNA (rRNA) isolated from the microbe Escherichia coli onto a DNA array. For the hybridization adsorption of both DNA and RNA oligonucleotides, a detection limit of 10 nM is reported; for large (1500-base) 16S rRNA molecules, concentrations as low as 2 nM are detected. The covalent attachment of thiol-DNA probes to the gold surface leads to high surface probe density (1012 molecules/cm2) and excellent probe stability that enables more than 25 cycles of hybridization and denaturing without loss in signal or specificity. Fresnel calculations are used to show that changes in percent reflectivity as measured by SPR imaging are linear with respect to surface coverage of adsorbed DNA oligonucleotides. Data from SPR imaging is used to construct a quantitative adsorption isotherm of the hybridization adsorption on a surface. DNA and RNA 18-mer oligonucleotide hybridization adsorption is found to follow a Langmuir isotherm with an adsorption coefficient of 1.8 × 107 M-1.