DNA-based molecular recognition system for lactoferrin biosensing

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](4)TAGAGGATCAAA3' 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: k(a) = (2.49 +/- 0.03)center dot 10(4) M-1 center dot s(-1), k(d) = (1.89 +/- 0.02)center dot 10(-3) s(-1), K-A = (0.13 +/- 0.05)center dot 10(8) M-1, and K-D = (7.61 +/- 0.18)center dot 10(-8) M. Thermodynamic study conducted to determine the Delta H-0, Delta S-0, and Delta G(0) 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 center dot 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.