Introduction 1 2 3 4 5 6 7 8 9 14 10 15 2 16 6 7 17 18 7 19 20 Materials and methods Materials 4 Synthesis Fluorescein-labeled estradiol derivative (E2-F) 7 19 1 1  tert 4 2 n 2 3 3 2 4 3 4 5 5 6  tert 2 6 7 7 18 8 1 3 J J J J J J J J J J J J J m/z + Fig. 1 Experimental  7-Hydroxy-3-p-hydroxyphenylcoumarine (coumarol) 21 p p 1 6 J J J J J m/z + Biomaterials Microsomes 22 Estrogen receptor α Escherichia coli 23 B max 24 B max Microsomal incubations 2 4 2 Instrumentation + 2 λ ex  λ em 20 Coumestrol-based microplate reader assay of ERα affinity 6 FP-based microplate reader assay of ERα affinity 2 FP-based ERα receptor affinity detection (RAD) in flow-injection analysis (FIA) mode 2 Fig. 2  S  FP-based ERα receptor affinity detection coupled on-line to gradient HPLC 14 2 2 λ ex  λ em Results and discussion 20 50 Synthesis of E2-F 1 2 3 7 7 19 19 6 50  K D 7 19 FP-based microplate reader assay of ERα affinity 3  K D 19 7  K D 50 Fig. 3 FP-based microplate reader tracer concentration optimization. Incubations in the presence of ERα ligand E2 (33.3 μM; equal to 100% tracer displacement) were compared with incubations without E2 (equal to 0% tracer displacement) at decreasing E2-F tracer concentrations  25 4 10 Fig. 4 FP-based microplate reader solvent optimization. Incubations in the presence of ERα ligand E2 (33.3 μM; equal to 100 % tracer displacement) were compared with incubations without E2 (equal to 0% displacement) at increasing MeOH, ACN and iPrOH concentrations at an E2-F concentration of 83.3 nM  FP-based ERα receptor affinity detection (RAD) in flow-injection analysis (FIA) mode 2 ERα LBD and E2-F tracer optimization 5  K D R max 1  K D 19 7  R max Fig. 5 K D R max  Table 1 K D R max [ERα] (nM) K D R max a 0.7 43.6 ± 12.4 6 1.5 26.4 ± 5.0 6 3.0 34.5 ± 5.3 6 a R max Organic modifier concentration 6 10 Fig. 6 Effect of MeOH and ACN reaction coil concentrations upon the assay performance in FIA mode. The S/N ratios of the E2 injections equalling 100% tracer displacement are displayed  Excitation and emission wavelengths Competitive receptor binding curves 50 50 2 7 26 50 Table 2 50 Compound 50  n  50  n  50 DES −9 −9 −9 a Tamoxifen −7 −7 −7 a Coumestrol −7 −7 −7 b E2 −9 −9 −9 a EE2 −9 −9 −9 b 50 a 7 b 26  DES  E2  EE2 FP-based ERα receptor affinity detection coupled on-line to HPLC 10 7 Fig. 7 1 2 3 4 5  8  t  t m/z m/z m/z m/z m/z 27 28 Fig. 8a–b a  M1  M2  M3 b  20