Label-free evaluation of small-molecule–protein interaction using magnetic capture and electrochemical detection
The evaluation of interaction between small molecules and protein is an important step in the discovery of new drugs and to study complex biological systems. In this work, an alternative method was presented to evaluate small-molecule–protein interaction by using ligand capture by protein-coated magnetic particles (MPs) and disposable electrochemical cells. The interaction study was conducted using -gingerol from ginger rhizome and a transmembrane protein αVβ3 integrin. Initially, the electrochemical behavior of the natural compound -gingerol was evaluated with the disposable carbon-based electrodes and presented an irreversible oxidation process controlled by diffusion. The analytical curve for -gingerol was obtained in the range of 1.0 to 20.0 μmol L−1, with limit of detection of 0.26 μmol L−1. Then MPs coated with αVβ3 integrin were incubated with standard solutions and extracts of ginger rhizome for -gingerol capture and separation. The bioconjugate obtained was dropped to the disposable electrochemical cells, keeping a permanent magnet behind the working electrode, and the binding process was evaluated by the electrochemical detection of -gingerol. The assay method proposed was also employed to calculate the -gingerol–αVβ3 integrin association constant, which was calculated as 4.3 × 107 M−1. The method proposed proved to be a good label-free alternative to ligand–protein interaction studies.
KeywordsLigand–protein binding assay Small-molecule–protein binding Magnetic capture Affinity constant
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, No. 150569/2016-5), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, No. 2015/19890-1, No. 2014/22401-0, and No. 2015/24940-8), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, No. 01).
Compliance with ethical standards
The authors declare that they have no competing interest.
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