The paper describes the development of a novel DNA oligonucleotide-based affinity bioreceptor that binds to lactoferrin, a glycoprotein-type immunomodulator. The research was performed using surface plasmon resonance method to investigate affinity of various types of oligonucleotides to the target protein. The 72 base pair-long 5'[(TAGAGGATCAAA)AAA]4TAGAGGATCAAA3' sequence with the highest affinity to lactoferrin was selected for further investigations. Kinetic analysis of the interaction between selected DNA and lactoferrin provided rate and equilibrium constants: ka = (2.49 ± 0.03)∙104 M-1∙s-1, kd = (1.89 ± 0.02)∙10-3 s-1, KA = (0.13 ± 0.05)∙108 M-1, and KD = (7.61 ± 0.18)∙10-8 M. Thermodynamic study conducted to determine the ΔH0, ΔS0, and ΔG0 for van't Hoff characteristic in the temperature range of 291.15-305.15 K, revealed the complex formation as endothermic and entropically driven. The chosen DNA sequence's selectivity towards lactoferrin was confirmed with interferents' response constituting <3 % of the response to lactoferrin. SPR analysis justified utility of the designed DNA oligonucleotide for Lf determination, with LOD of 4.42∙10-9 M. Finally, the interaction between lactoferrin and DNA was confirmed by electrochemical impedance spectroscopy, providing the basis for further quantitative assay of lactoferrin using the developed DNA-based bioreceptor. The interactions were performed under immobilized DNA ligand conditions, thus reflecting the sensor's surface, which facilitates their transfer to other label-free biosensor technologies.
Keywords: DNA–protein interaction; Lactoferrin; Surface plasmon resonance.
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