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Non-Langmuir Kinetics of DNA Surface Hybridization.

ABSTRACT: Hybridization of complementary single strands of DNA represents a very effective natural molecular recognition process widely exploited for diagnostic, biotechnology, and nanotechnology applications. A common approach relies on the immobilization on a surface of single-stranded DNA probes that bind complementary targets in solution. However, despite the deep knowledge on DNA interactions in bulk solution, the modeling of the same interactions on a surface are still challenging and perceived as strongly system dependent. Here, we show that a two-dimensional analysis of the kinetics of hybridization, performed at different target concentrations and probe surface densities by a label-free optical biosensor, reveals peculiar features inconsistent with an ideal Langmuir-like behavior. We propose a simple non-Langmuir kinetic model accounting for an enhanced electrostatic repulsion originating from the surface immobilization of nucleic acids and for steric hindrance close to full hybridization of the surface probes. The analysis of the kinetic data by the model enables quantifying the repulsive potential at the surface, as well as retrieving the kinetic parameters of isolated probes. We show that the strength and the kinetics of hybridization at large probe density can be improved by a three-dimensional immobilization strategy of probe strands with a double-stranded linker.

SUBMITTER: Vanjur L

PROVIDER: S-EPMC7474173 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Milk polar lipid interfacial behavior continues to be analyzed using Langmuir trough experiments, but the reported Langmuir trough subphases commonly used are not fully representative of milk. A method to transform liquids of biological origin, such as milk, into appropriate Langmuir trough subphases does not currently exist, which hinders the applicability of Langmuir trough experiments to nature. Here, a procedure to manufacture milk-derived Langmuir trough subphases with insignificant amounts of surfactants from bovine milk is presented. Ultrafiltration is used to remove the bulk of protein surfactants from milk followed by the creation of solvent-induced emulsions that remove trace proteins and lipids from collected skim milk permeates. Change in surface tension upon compression of resulting washed permeates from the surfactant removal process was ≤0.1 mN/m, terming the permeates non-surface-active (NSA). NSA permeates (72.4 ± 0.2 mN/m) had a surface tension similar to that of ultrapure water (72.6 ± 0.1 mN/m), but their pH, conductivity, percent total solids, Brix percentage, alkalinity, and hardness were not the same, with NSA permeates being more compositionally similar to skim milk than water. The lift-off points of milk ganglioside GM3 monolayer surface pressure-area isotherms spread on NSA permeates and ultrapure water subphases were significantly different when compared for the same sample, indicating that surface tension measurements obtained on ultrapure water are not the same as with NSA milk permeate. Overall, surfactants were removed from bovine milk without the addition of exogenous compounds, allowing for the production of a NSA solution derived from milk that can be used in the Langmuir trough experiment to more realistically resemble the natural environment of milk polar lipids. The procedure described here was able to produce NSA solutions for other dairy beverages aside from milk, indicating that it can be applicable to other biological fluids.

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Non-Langmuir Kinetics of DNA Surface Hybridization.

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