乳铁蛋白
寡核苷酸
表面等离子共振
DNA
化学
生物传感器
杂交探针
费斯特共振能量转移
分析化学(期刊)
生物化学
色谱法
纳米技术
材料科学
荧光
纳米颗粒
物理
量子力学
作者
Agnieszka Paziewska−Nowak,Marcin Urbanowicz,Kamila Sadowska,D. G. Pijanowska
标识
DOI:10.1016/j.ijbiomac.2023.126747
摘要
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.
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