Optical Study of DNA Surface Hybridization Reveals DNA Surface Density as a Key Parameter for Microarray Hybridization Kinetics

Abstract

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We discuss the implications with respect to DNA microarray detection.

Label-free quantitative DNA detection using the liquid core optical ring resonator

“…6 shows the net WGM shift in response to the target DNA concentration, which can be easily modeled as a function of bulk concentration using a form of Michaelis-Menton: where ␦λ WGM is the relative spectral shift, ␦λ max is the maximum possible shift observed for maximum surface coverage with target, K d is the dissociation constant for hybridization, and [DNA] is the bulk concentration of the target strand. A Michaelis-Menton fit produces a dissociation constant K d of 2.9 nM, close to what has been reported in previous studies (Lehr et al, 2003;Michel et al, 2007). ␦λ max is 5.1 pm, which is half of the saturation WGM shift when a higher probe density was used.…”

Kinetic Mechanisms in Morpholino–DNA Surface Hybridization

“…That consumption of the probe reagent was not accompanied by slowing of the reaction is suggestive of an “autocatalytic” effect. Our inspection of published hybridization traces reveals that such behavior can also arise in hybridization to PNA probes (especially at lower ionic strengths) 53-54 and, at times, to DNA probes, 26,55 as well as in protein adsorption. 56 At even longer times, stage III in Fig.…”

Stability of a Surface-Bound Oligonucleotide Duplex Inferred from Molecular Dynamics: A Study of Single Nucleotide Defects Using DNA Microarrays

“…When the temperature of a solution containing double stranded DNA is raised above the melting temperature, it reversibly separates into two single strands (of complementary sequence). The reverse reaction, termed hybridization [1], is reduced if noncomplementary (mismatched) bases are present [2]. The stability of the DNA duplex is due to hydrogen bonding of the complementary pairs, as well as base stacking interactions between adjacent base pairs, which include van der Waals, electrostatic, and hydrophobic interactions.…”