Method to screen aromatic ligands in mixtures for quantitative affinities to target using magnetic separation of bound ligands along with HPLC and UV photometry detection

• Published in: Mikrochimica acta (1966) Vol. 176; no. 1-2; pp. 243 - 249
• Main Authors: Yang, Xiaolan, Pu, Jun, Zhao, Hua, Li, Xiaoyan, Liao, Juan, Xie, Yanling, Zhu, Sha, Long, Gaobo, Yuan, Yonghua, Liao, Fei
• Format: Journal Article
• Language: English
• Published: Vienna Springer Vienna 2012; Springer
• Subjects: Analysis; Analytical Chemistry; Biotin; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatographic methods and physical methods associated with chromatography; Exact sciences and technology; High performance liquid chromatography; Medical screening; Methods; Microengineering; Nanochemistry; Nanotechnology; Other chromatographic methods; Short Communication; Spectrometric and optical methods; Relative affinity; Magnetic separation; HPLC-UV; Parallel-combinatorial-syntheses; Biotin derivatives; Streptavidin; Purification; Ligand; Use; HPLC chromatography; Immobilization; Biotin; Ultraviolet spectrometry; Screening test; Mixture; Protein; Screening; Target; Ultraviolet detector; Test method; Affinity
• ISSN: 0026-3672; 1436-5073
• DOI: 10.1007/s00604-011-0696-y

A new screening method was tested to estimate the affinity of an aromatic ligand in a mixture through competitive binding against an exogenous reference ligand. The target protein was immobilized on magnetic particles and one or several ligand(s) in each reaction mixture were prepared by parallel combinatorial-synthesis without purification. Specifically, the binding of aromatic biotin derivatives to streptavidin immobilized on magnetic particles was used as the model. An approximation equation that works under the condition of binding ratios below 0.1 for the candidate ligand and the reference ligand was developed. It was found that the relative affinity of each biotinylated aromatic candidate ligand in mixtures was consistent with that by using HPLC-MS analyses or by a homogenous method using its purified counterpart. Hence, this new screening method using HPLC-UV is considered to be highly effective. Figure The representative procedure to realize the new screening technique for quantitative affinity of an aromatic candidate ligand of unknown quantity in a mixture sample