Journal
MICROCHIMICA ACTA
Volume 176, Issue 1-2, Pages 243-249Publisher
SPRINGER WIEN
DOI: 10.1007/s00604-011-0696-y
Keywords
Relative affinity; HPLC-UV; Magnetic separation; Biotin derivatives; Streptavidin; Parallelcombinatorial-syntheses
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Funding
- Program for New Century Excellent Talent in University [NCET-09]
- National Natural Science Foundation of China [30472139, 30672009, 81071427]
- Natural Science Foundation of CQ [CSTC2011BA5039]
- Chongqing Education Commission [KJ100313]
- Chongqing Medical University [2002CX03]
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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.
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