Effect of Probe–Probe Distance on the Stability of DNA Hybrids on Surfaces

We have used temperature gradient surface plasmon resonance (SPR) measurements to quantitatively evaluate how the stability of different types of hybrids formed with DNA probes on surfaces is affected by probe spacing. SPR sensors with different average surface densities of probes were prepared by coadsorbing probes with lateral spacers strands comprised of phosphorothioated adenine nucleotides (A15*). Increasing the fraction of A15* spacers in the immobilization solution results in larger distances between probes on the sensor, determined here using a combination of SPR and X-ray photoelectron spectroscopy (XPS) measurements. The hybridization activities of probes were simultaneously measured over a temperature range that spanned the denaturation temperature ( T m ) of hybrids by applying a spatial temperature gradient across the sensor surface. The resulting temperature profiles of hybridization activity show how the stability of hybrids increases as either the distance between probes or the ionic strength of the hybridization buffer increase. Additionally, hybridization activity profiles sharpen as the spacing between probes increases, indicating more homogeneous hybridization behavior of probes. The results provide quantitative experimental data for testing theoretical models of stability, supporting models that account for both repulsive interactions between DNA strands and local variability in probe surface density.