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Issue 11, 2012
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Development of a label-free competitive ligand binding assay with human serum albumin on a molecularly engineered surface plasmon resonance sensor chip

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Abstract

The binding interactions between ligands and receptors play a vital role in their biological functions. In this work, a label-free indirect competitive binding assay based on surface plasmon resonance (SPR) detection was developed, using fatty acid–human serum albumin (HSA) as a model system. In the assay, a fatty acid analog was immobilized on the surface of an SPR gold chip by self-assembly, and was allowed to bind with HSA in solution. Addition of a ligand under investigation into the solution set up a competition with the analog for the protein, causing a reduction in the SPR signal. Due to the asymmetric structure of the ligand binding channel, HSA exhibited remarkable selectivity for the orientation of the fatty acid analog on a gold surface. When the carboxylic acid group of the analog was exposed on the sensor surface, the amount of bound HSA was very low. However, it increased by more than 7 fold when the alkyl chain was exposed. Dilution of the surface coverage of the fatty acid analog also enhanced HSA binding by reducing steric hindrance on the surface. Using a 1 : 4 mercaptoundecanoic acidmercaptohexanol modified surface derivatized with octylamine, competitive binding assays for octanoic acid and perfluorooctane sulfonate with HSA were conducted. Using a derived kinetic equilibrium model, the equilibrium association constant was determined to be 4.57 × 105 M−1 and 2.11 × 105 M−1, respectively, which are consistent with the values measured by other established methods. The engineered sensor chip surface can be used to study the binding interactions of other chemicals with this important serum transport protein.

Graphical abstract: Development of a label-free competitive ligand binding assay with human serum albumin on a molecularly engineered surface plasmon resonance sensor chip

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Publication details

The article was received on 20 Jul 2012, accepted on 28 Aug 2012 and first published on 12 Sep 2012


Article type: Paper
DOI: 10.1039/C2AY25780G
Citation: Anal. Methods, 2012,4, 3718-3723
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    Development of a label-free competitive ligand binding assay with human serum albumin on a molecularly engineered surface plasmon resonance sensor chip

    Y. Gao, X. Li and L. Guo, Anal. Methods, 2012, 4, 3718
    DOI: 10.1039/C2AY25780G

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