The effect of surface probe density on DNA hybridization

TLDR

Hybridization of complementary DNA strands underlies all microarray‑based DNA variation analyses. The study investigates how surface probe density influences target capture kinetics measured by surface plasmon resonance spectroscopy. Probe density was varied by altering immobilization conditions such as ionic strength, interfacial electrostatic potential, and the use of duplex versus single‑stranded oligonucleotides. DNA films of equal probe density exhibit reproducible hybridization efficiencies and kinetics, yet overall efficiency and capture rates vary strongly with probe density, indicating density effects account for previously attributed thermodynamic variations.

Abstract

The hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. In this paper, we study how probe immobilization at surfaces, specifically probe density, influences the kinetics of target capture using surface plasmon resonance (SPR) spectroscopy, an in situ label-free optical method. Probe density is controlled by varying immobilization conditions, including solution ionic strength, interfacial electrostatic potential and whether duplex or single stranded oligonucleotides are used. Independent of which probe immobilization strategy is used, we find that DNA films of equal probe density exhibit reproducible efficiencies and reproducible kinetics for probe/target hybridization. However, hybridization depends strongly on probe density in both the efficiency of duplex formation and the kinetics of target capture. We propose that probe density effects may account for the observed variation in target-capture rates, which have previously been attributed to thermodynamic effects.