Electrostatic repulsion and steric hindrance effects of surface probe density on deoxyribonucleic acid (DNA)/peptide nucleic acid (PNA) hybridization

Abstract In this paper, we study how probe density influences the kinetics of target capture by surface-immobilized probe strands in oligonucleotide hybridization. Surface plasmon field-enhanced fluorescence spectroscopy was employed to investigate the hybridization behaviors of natural DNA or synthetic peptide nucleic acid (PNA) double strands and quantitatively characterize surface probe density effects. Two kinds of interfacial architectures are used for the study: a self-assembled thiol/streptavidin layer (planar matrix) and a colloidal streptavidin–gold conjugate particles layer (particle matrix) coupled to a flat Au surface. For both matrices, the hybridization reactions were investigated as a function of probe density. Surface effects, including electrostatic repulsion between double stranded DNA hybrids, repulsion of negatively charged free DNA molecules in the bulk by the DNA immobilized at the surface, and steric hindrance effects, were studied. The study reveals that a monotonic decrease of the affinity constant for DNA hybridization was observed for both matrices with increasing probe density, which was attributed to steric hindrance and electrostatic repulsion. Owing to the neutral backbone of PNA, the hybridization reaction was only influenced by the variation of steric hindrance effects. The dilution of the PNA probe surface layer significantly reduced this steric hindrance and enhanced the hybridization affinity.