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Abstract: Antibody mimics have been prepared by molecular imprinting of (S)- propranolol and applied in the development of radioligand binding assays for the imprint species. In the assays, polymer particles, radioligand, and analyte were incubated either in an organic solvent or an aqueous buffer, with similarly high sensitivity under both set of conditions, Optimization of the assay conditions led to 100-1000- fold improvements in the limit of determination and 100-1000-fold reductions of the amount of imprinted polymer used, compared with previous studies of this novel type of assay. The present assay uses only 10-50 mu g of polymer, and the limit of determination is about 6 nM. The toluene-based assay showed excellent enantioselectivity, the cross-reactivity of the R enantiomer being only 1%, which is better than that demonstrated by biological antibodies. The aqueous buffer based assay showed high substrate selectivity for propranolol in the presence of structurally similar beta-blockers. The different selectivity profiles obtained are due to a different balance between hydrophobic and polar interactions in toluene and water, since polar interactions, such as hydrogen bonds, are strong in apolar solvents and hydrophobic interactions are strong in water