Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization We describe the development and validation of a high-resolution screening -LRB- HRS -RRB- platform which couples gradient reversed-phase high-performance liquid chromatography -LRB- RP-HPLC -RRB- on-line to estrogen receptor α -LRB- ERα -RRB- affinity detection using fluorescence polarization -LRB- FP -RRB- . FP , which allows detection at high wavelengths , limits the occurrence of interference from the autofluorescence of test compounds in the bioassay . A fluorescein-labeled estradiol derivative -LRB- E2-F -RRB- was synthesized and a binding assay was optimized in platereader format . After subsequent optimization in flow-injection analysis -LRB- FIA -RRB- mode , the optimized parameters were translated to the on-line HRS bioassay . Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic -LRB- 17β-estradiol , 17α-ethinylestradiol and the phytoestrogens coumestrol , coumarol and zearalenone -RRB- , followed by post-column bioaffinity screening of the individual affinities for ERα . Using the HRS-based FP setup , we were able to screen affinities of off-line-generated metabolites of zearalenone for ERα . It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence . Introduction In recent years , the presence of endocrine-disrupting compounds -LRB- EDCs -RRB- in the environment has become a major issue of concern in view of their effects on both human health and ecosystem integrity . The classes of substances that possess estrogenic activities include diverse manmade and natural chemicals -LRB- e.g. , pesticides , plasticizers , polychlorinated biphenyls , phytoestrogens , natural hormones -RRB- and pharmaceuticals -LRB- e.g. , ethinylestradiol , tamoxifen -RRB- -LSB- 1 , 2 -RSB- . EDCs can interfere with the endocrine system by binding to the estrogen receptors α -LRB- ERα -RRB- or β -LRB- ERβ -RRB- and mimicking the action of the naturally produced hormone estrogen or blocking the receptor for this hormone in target cells , thereby preventing its action -LSB- 3 -RSB- . Therefore , it is important to screen compounds and compound mixtures for their ability to bind to one of these receptors . Alternatively , the task of screening for compounds containing estrogenic and antiestrogenic properties is also very important from medical and pharmaceutical points of view -LSB- 4 -RSB- . Several biodetection methodologies have meanwhile been developed to determine the bioaffinities of ligands towards ERα . These assays include relatively cumbersome radioligand binding assays -LSB- 5 -RSB- , fluorescence enhancement based assays -LSB- 6 -RSB- and fluorescence polarization -LRB- FP -RRB- based binding assays -LSB- 7 -RSB- . A major drawback of these assays is that , in the presence of cross-reactive compounds , the response provided by the bioassay reflects the sum of the concentrations of all individual compounds and their affinities for the ERα . Moreover , matrix components , which are sometimes present in far higher concentrations than the analyte -LRB- s -RRB- , interfere with the binding assay signals , thereby leading to false-positive or false-negative results -LSB- 8 -RSB- . Therefore , none of these methods can be applied for the identification of individual ERα binding ligands in mixtures of compounds , as is the case with natural compound extracts , biological samples containing different EDCs , and drug metabolite mixtures . However , the introduction of the on-line high-resolution screening -LRB- HRS -RRB- technology enabled the screening of individual compounds in mixtures by coupling a separation technology , usually gradient HPLC , to post-column biochemical detection assays on-line -LSB- 9 -- 14 -RSB- . Oosterkamp et al. -LSB- 10 -RSB- developed a HRS system to screen mixtures for compounds with an affinity for ERα . A similar setup has been used to screen natural plant product extracts for ERα and ERβ binding activity -LSB- 15 -RSB- and to identify and screen cytochrome P450-derived metabolites of tamoxifen with ERα affinity -LSB- 2 , 16 -RSB- . The reported HRS ERα bioaffinity assays are based on the increase of the fluorescent signal of the tracer compound coumestrol upon binding to the ERα ligand binding domain -LRB- LBD -RRB- . When the tracer is displaced by a compound with ERα affinity that elutes from the HPLC column , it is seen as a negative peak in the baseline of the ERα bioassay . Coumestrol , the fluorescent probe , has short excitation -LRB- 340 nm -RRB- and emission -LRB- 410 nm -RRB- wavelengths when bound to the ERα LBD -LSB- 6 -RSB- . Since there are many potentially interfering fluorescent compounds that can be excited at 300 -- 350 nm and emit at 360 -- 430 nm in biomatrices , coumestrol is not appropriate for the precise and selective detection of EDCs in biosamples containing phytoestrogens such as flavonoids , coumestans or lignans . One solution to this problem is to use another tracer compound , thus making it possible to monitor the bioassay signal at higher excitation and emission wavelengths . However , no tracer compounds are currently available which also display the necessary fluorescence enhancement upon binding to the ERα , thus making it impossible to use them in a HRS ERα setup . Another option is fluorescence polarization -LRB- FP -RRB- detection . FP is based upon the principle that interaction of a receptor with a fluorophore-containing ligand increases the effective size of the fluorophore through the formation of a receptor ligand complex . The increase in effective size reduces the rate of fluorophore rotation and increases the polarization over that of the unbound fluorophore -LSB- 7 -RSB- . FP instruments measure the rate of fluorophore rotation in solution rather than fluorescence intensity -LSB- 17 -RSB- . The polarization value is independent of the fluorophore concentration and intensity and has the advantage that it is minimally affected by autofluorescence , solution opacity or color , and is more tolerant to fluorescence quenching and light scattering -LSB- 18 -RSB- . The objective of the present study was to develop and evaluate the use of FP detection in combination with the on-line HRS technology to provide information about the ERα affinities of individual compounds in complex mixtures . The assay is based on competition for the ERα LBD between eluting compounds and a fluorescein-labeled estradiol derivative -LRB- E2-F -RRB- , which was synthesized based on tracer structures described by Parker et al. -LSB- 7 -RSB- and Ohno et al. -LSB- 19 -RSB- for off-line FP-based ER assays . After synthesis of the tracer E2-F , the ERα binding assay was first optimized in FP platereader format and subsequently transferred to flow-injection analysis -LRB- FIA -RRB- mode with FP detection by using an on-line FP detector cell , which was recently constructed by Kool et al. -LSB- 20 -RSB- to measure GPCR-mediated modulation of cAMP production . Finally , after optimization of the assay in FIA mode , the on-line FP detection system was coupled to a gradient HPLC system . In this so-called HRS setup we separated mixtures of known estrogenic compounds -- e.g. , 17β-estradiol , 17α-ethinylestradiol and the phytoestrogens coumestrol , coumarol and zearalenone -- and screened the individual compounds for ERα affinities . We also showed that this HRS ERα-FP system was suitable for screening off-line-generated cytochrome P450 -LRB- Cyt P450 -RRB- metabolites of zearalenone for ERα affinity . We conclude that we were able to incorporate an on-line FP detector cell into a HRS system to screen for the ERα affinities of individual compounds in complex mixtures without incurring interference from the autofluorescence of the compounds . Materials and methods Materials Tween 20 , 17β-estradiol -LRB- E2 -RRB- , estrone -LRB- E1 -RRB- , 17α-ethinyl estradiol -LRB- EE2 -RRB- , diethylstilbestrol -LRB- DES -RRB- , estriol , zearalenone , fluorescein-5-isothiocyanate -LRB- 5-FITC -RRB- and coumestrol were obtained from Sigma-Aldrich -LRB- Zwijndrecht , The Netherlands -RRB- . ELISA blocking reagent was purchased from Roche -LRB- Mannheim , Germany -RRB- . Dry THF was freshly distilled from LiAlH4 . All other chemicals were of analytical grade and obtained from standard suppliers . Synthesis Fluorescein-labeled estradiol derivative -LRB- E2-F -RRB- The fluorescein-labeled estradiol derivative -LRB- E2-F -RRB- was synthesized in analogy to methods described previously -LSB- 7 , 19 -RSB- from estrone as the starting material -LRB- Fig. 1 -RRB- . To a solution of estrone -LRB- 1 , 700 mg , 2.59 mmol -RRB- in dry DMF -LRB- 3 mL -RRB- , imidazole -LRB- 250 mg -RRB- was added . Subsequently , tert-butyl dimethylsilyl chloride -LRB- tBDSC -RRB- dissolved in dry THF -LRB- 2 mL -RRB- was slowly added and the reaction mixture was stirred at room temperature for 3 h . The resulting cloudy white mixture was extracted with ethyl acetate -LRB- EtOAc -RRB- . The organic extract was washed with water once , with brine twice and then with water once again . The organic extract was dried over MgSO4 , filtered , and concentrated by rotary evaporation at 45 °C . Recrystallization was performed using a mixture of 15 mL MeOH and 5 mL DCM to afford 2 -LRB- 667.2 mg , 67 % -RRB- . Under an atmosphere of dry nitrogen , 3-butyn-1-ol -LRB- 1.35 g -RRB- was dissolved in dry THF -LRB- 27 mL -RRB- and cooled to − 78 °C . n-Butyllithium -LRB- BuLi -RRB- in hexane -LRB- 16.9 mL , 0.027 mol -RRB- was added dropwise at − 78 °C . Compound 2 -LRB- 3.60 g , 9.36 mmol -RRB- dissolved in dry THF -LRB- 25 mL -RRB- was carefully added dropwise at − 78 °C and the solution was allowed to warm to room temperature and was thereafter stirred at 40 °C for an additional 60 min . The remaining BuLi was neutralized by carefully adding 5 mL EtOAc/MeOH -LRB- 1:1 -RRB- . Extraction was performed with EtOAc , as described above . The crude product was purified by column chromatography on silica gel -LRB- 10 % EtOAc in hexane -RRB- to afford 3 -LRB- 1.62 g , 38 % -RRB- . To a solution of 3 -LRB- 1.6 g , 3.52 mmol -RRB- dissolved in dry THF -LRB- 25 mL -RRB- was added triethylamine -LRB- TEA , 718 μL -RRB- followed by MeSO2Cl -LRB- 395 μL , 5.1 mmol -RRB- while stirring at 0 °C . Afterwards the solution was allowed to warm to room temperature . After 10 h the reaction mixture was extracted with DCM . The organic extract was treated as described above and concentrated by rotary evaporation to afford 4 -LRB- 1.80 g , 96 % -RRB- . Under an atmosphere of dry nitrogen , NaN3 -LRB- 8.0 g -RRB- was added to a solution of 4 -LRB- 1.80 g , 3.38 mmol -RRB- dissolved in dry DMPU -LRB- 25 mL -RRB- while stirring rapidly . After stirring the solution for three days , the resulting reaction mixture was extracted with EtOAc as described above to afford 5 -LRB- 1.36 g , 84 % -RRB- . To a solution of 5 -LRB- 1.36 g , 2.83 mmol -RRB- in MeOH -LRB- 70 mL -RRB- and TEA -LRB- 16 mL -RRB- , 1,3-propane dithiol -LRB- 12 mL -RRB- was added . The solution was stirred o/n at room temperature and was concentrated by rotary evaporation . The concentrated solution dissolved in 100 mL hexane was purified by column chromatography on silica gel . Elution of the purified product 6 -LRB- 1.08 g , 84 % -RRB- was performed using EtOAc/MeOH -LRB- 1:1 -RRB- . To remove the protective tert-butyl dimethylsilyl group , TBAF.H2O -LRB- 300 mg -RRB- was added to a solution of 6 -LRB- 258 mg , 0.57 mmol -RRB- dissolved in dry THF -LRB- 4 mL -RRB- at 0 °C . After stirring for 1 h and allowing the solution to warm to room temperature , the crude product was extracted with DCM . The product was purified with Flash Master chromatography with an EtOAc/MeOH gradient to afford 7 -LRB- 176 mg , 91 % -RRB- . Fluorescein-5-isothiocyanate -LRB- 5-FITC , 7 mg , 17.8 μmol -RRB- was added to a solution of 7 -LRB- 5 mg , 14.7 μmol -RRB- dissolved in dry DMF -LRB- 500 μL -RRB- and dry pyridine -LRB- 25 μL -RRB- . The solution was stirred o/n in the dark at room temperature . The crude product was extracted with DCM and purified first with Flash Master chromatography -LRB- silicagel-based ; EtOAC/MeOH gradient -RRB- and then with preparative C18 HPLC to afford 8 -LRB- E2-F , 8.02 mg , 75 % -RRB- . 1H NMR -LRB- 400.13 MHz , CD3OD -RRB- : δ 8.04 -LRB- 1 H , d , J = 1.9 Hz -RRB- , 7.70 -LRB- 1 H , dd , J = 8.2 , 1.9 Hz -RRB- , 7.10 -LRB- 1 H , d , J = 8.2 Hz -RRB- , 7.01 -LRB- 1 H , d , J = 8.5 Hz -RRB- , 6.89 -LRB- 1 H , d , J = 8.9 Hz -RRB- , 6.88 -LRB- 1 H , d , J = 8.9 Hz -RRB- , 6.66 -LRB- 2 H , d , J = 2.3 Hz -RRB- , 6.58 -LRB- 2 H , dd , J = 8.9 , 1.9 Hz -RRB- , 6.47 -LRB- 1 H , dd , J = 8.5 , 2.6 Hz -RRB- , 6.42 -LRB- 1 H , d , J = 2.6 Hz -RRB- , 3.80 -LRB- 2 H , t , J = 6.6 Hz -RRB- , 2.70 -LRB- 1 H , d , J = 1.9 Hz -RRB- , 2.65 -LRB- 2 H , t , J = 6.6 Hz -RRB- , 2.1 -- 2.3 -LRB- 3 H , m -RRB- , 1.9 -- 2.0 -LRB- 2 H , m -RRB- , 1.65 -- 1.85 -LRB- 4 H , m -RRB- , 1.25 -- 1.50 -LRB- 5 H , m -RRB- , 0.85 -LRB- 3 H , s -RRB- . ESI-MS : m/z 729 -LRB- M + H -RRB- + . Fig. 1Synthesis of a fluorescein-labeled estradiol -LRB- E2-F -RRB- derivative -LRB- for abbreviations , see `` Experimental '' section -RRB- 7-Hydroxy-3-p-hydroxyphenylcoumarine -LRB- coumarol -RRB- The synthesis of coumarol was analogous to the synthesis of benzene-hydroxylated-3-aryl-coumarins described by Buu-Hoi et al. -LSB- 21 -RSB- . Briefly , 2,4-dimethoxybenzaldehyde was reacted with p-methoxyphenyl-acetonitrile to form α-p-methoxyphenyl-β - -LRB- 2,4-dimethoxyphenyl -RRB- - acrylonitrile . The formed product formed was demethylated using pyridine hydrochloride to form the final product coumarol . 1H NMR -LRB- 400.13 MHz , DMSO-d6 -RRB- : δ 10.57 -LRB- 1 H , s -RRB- , 9.68 -LRB- 1 H , s -RRB- , 8.00 -LRB- 1 H , s -RRB- , 7.56 -LRB- 1 H , d , J = 8.6 Hz -RRB- , 7.53 -LRB- 2 H , d , J = 8.7 Hz -RRB- , 6.81 -LRB- 2 H , d , J = 8.7 Hz -RRB- , 6.80 -LRB- 1 H , dd , J = 10.8 , 2.7 Hz -RRB- , 6.73 -LRB- 1 H , d , J = 2.2 Hz -RRB- . ESI-MS : m/z 255 -LRB- M + H -RRB- + . Biomaterials Microsomes Rat liver microsomes were prepared from phenobarbital -LRB- PB -RRB- - induced rat liver , as described previously by Roosenboom et al. -LSB- 22 -RSB- . Estrogen receptor α The ERα ligand binding domain -LRB- LBD -RRB- was expressed in Escherichia coli according to the method of Eiler et al. -LSB- 23 -RSB- , but without estradiol in the medium . The concentration of ERα LBD -LRB- 250 nM -RRB- was measured by determining the Bmax value -LRB- by titration with radiolabeled estradiol -RRB- -LSB- 24 -RSB- . The Bmax value was measured as the maximum amount of ligand binding extrapolated to a very high concentration of ligand . Microsomal incubations Microsomal PB-induced rat liver incubations were carried out in a total volume of 500 μL . The reaction mixture consisted of 2.5 mM MgCl2 in 100 mM potassium phosphate buffer -LRB- pH 7.4 -RRB- , 100 μM zearalenone and 2.6 mg/mL rat liver microsomes . The reactions were initiated by the addition of a NADPH regenerating system , resulting in final concentrations of 0.1 mM NADPH , 0.3 mM glucose-6-phosphate , and 0.4 U/mL glucose-6-phosphate dehydrogenase . Incubation was carried out at 37 °C for 120 min and the reaction was terminated by the addition of 50 μL 10 % HClO4 . Following centrifugation at 14,000 rpm for 10 min , 450 μL of supernatant was applied to a strata_X 33 μm Polymeric Sorbent 200 mg / 3 mL -LRB- Phenomenex , Amstelveen , The Netherlands -RRB- column for SPE sample preparation . Before sample application the column was conditioned with 2 mL MeOH and subsequently equilibrated with 2 mL H2O . The sample was washed with 2 mL 5 % -LRB- v/v -RRB- of aqueous methanol and elution was performed using 2 mL 50 % -LRB- v/v -RRB- of MeOH in ACN . After evaporation , the residues were dissolved in 1 mL 40 % -LRB- v/v -RRB- of aqueous methanol . Instrumentation A NOVOstar + microplate fluorometer from BMG LABTECH GmbH -LRB- Offenburg , Germany -RRB- was used for the FP-based microplate reader assay . The excitation wavelength was set at 485 nm , the emission wavelength at 520 nm . Black-bottomed PP-96-well microtiter plates from Greiner Bio-one -LRB- Alphen a/d Rijn , The Netherlands -RRB- were used for all microplate reader experiments . The setup we used was adapted from the homogeneous coumestrol-based ERα affinity detection system described by Kool et al. -LSB- 2 -RSB- . For the on-line FP assay , two Knauer K-500 HPLC pumps -LRB- Berlin , Germany -RRB- were used to control the 120-mL superloops , made in-house , containing receptor -LRB- ERα -RRB- and tracer ligand -LRB- E2-F -RRB- , respectively , and one Knauer K-500 HPLC pump was used to deliver the injected samples . The ERα and E2-F solutions were prepared in sodium phosphate buffer -LRB- 10 mM ; pH 7.4 -RRB- containing 150 mM NaCl and 0.4 mg/mL ELISA blocking reagent . Both superloops were kept on ice . Flow restrictors were inserted between the pumps and the superloops to ensure proper operation of the pumps at low flow rates . The flow restrictors consisted of natural PEEK tubing and resulted in back pressures of approximately 50 bar . The pressure limits of the pumps were set 20 bar higher than the working pressure , and VICI Jour backpressure regulators -LRB- Schenkon , Switzerland -RRB- were inserted after the superloops to prevent damage from possible clogging of the system . A Gilson 234 autoinjector -LRB- Villiers-le-Bel , France -RRB- equipped with a Rheodyne -LRB- Bensheim , Germany -RRB- six-port injection valve -LRB- different injection loops -RRB- was used to inject samples . To maintain the reaction coils at a constant temperature , a Shimadzu CTO-10AC column oven -LRB- Duisburg , Germany -RRB- was integrated into the system . An Agilent 1100 -LRB- Waldbronn , Germany -RRB- series fluorescence detector -LRB- λex 498 nm ; λem 522 nm -RRB- was used to monitor fluorescence polarization , as described by Kool et al. -LSB- 20 -RSB- . Briefly , the standard 8 μL FLD cell was modified with parallel-placed -LRB- both mounted in the detector cell -RRB- fluorescence polarization filters from Melles Griot -LRB- Didam , The Netherlands -RRB- . One filter -LRB- 12.5 mm visible pol . 350 -- 650 nm 03FPG019 -RRB- was placed in the pathway of the excitation beam and one -LRB- Mount . Sheet Pol . 20 . D - 16.9 CA 03FPG001 -RRB- in the pathway of the emission beam . All hardware was integrated into one HRS system by Kiadis B.V. -LRB- Groningen , the Netherlands -RRB- , and was controlled by software developed by Kiadis B.V. Coumestrol-based microplate reader assay of ERα affinity The microplate reader assay for determining ERα binding based on the competition between fluorescent coumestrol and estrogenic compounds previously described by Gurer-Orhan et al. -LSB- 6 -RSB- was used to plot a competitive binding curve for E2-F . FP-based microplate reader assay of ERα affinity The microplate reader assay was used to optimize the FP ERα receptor affinity detection -LRB- RAD -RRB- system in FIA and HPLC modes . This assay is based on the competition between estrogenic compounds and the newly synthesized fluorescein-labeled estradiol derivative -LRB- E2-F -RRB- for binding to the ERα LBD . Displacement of E2-F was measured as the decrease in fluorescence polarization intensity . Different concentrations of E2-F tracer were tested first . Since Tween 20 , a detergent also used by Kool et al. in their coumestrol-based HRS ERα assay -LSB- 2 -RSB- , can improve the resolution of the assay in HRS mode , and since organic modifiers are necessary for the optimal performance of the gradient HPLC system , the next step in the optimization process was to vary these parameters . The experiments were carried out at 24 °C with well volumes of 250 μL binding buffer -LSB- potassium phosphate buffer -LRB- 10 mM ; pH 7.4 -RRB- containing 0.4 mg/ml ELISA blocking reagent -RSB- . The ERα LBD was introduced into the binding assay buffer at a final concentration of 1.64 nM . The E2-F stock solution was prepared in DMSO and dilutions were made in binding buffer . Concentrations ranging from 0.42 nM to 3.31 μM were investigated for E2-F optimization . For the experiments to investigate the detergent and organic modifier effects , the final concentration of E2-F in the assay was 83.3 nM . Aliquots of 25 μL of organic modifier or detergent at various concentrations were pipetted into a 96-well plate that already contained either 75 μL binding buffer -LRB- equal to 0 % displacement -RRB- or 75 μL 33.3 μM E2 in binding buffer -LRB- equal to 100 % displacement -RRB- . The 96-well plate was gently but thoroughly mixed after pipetting 75 μL E2-F into each well . Finally , 75 μL of the ERα LBD was pipetted into the wells . After an incubation time of 60 min at room temperature , the fluorescence polarization -LRB- FP -RRB- was recorded at 485 nm excitation and 520 nm emission . All experiments were performed in quadruplicate . The difference in FP -LRB- ΔFP -RRB- was calculated by subtracting the FP value found at 100 % displacement from the FP value found when no E2 was present under different conditions . FP-based ERα receptor affinity detection -LRB- RAD -RRB- in flow-injection analysis -LRB- FIA -RRB- mode A schematic overview of the RAD system in FIA mode is depicted in Fig. 2 . In this system the flow from a superloop containing ERα LBD is continuously mixed with flow from a superloop containing E2-F . Compound samples -LRB- 10 μL -RRB- are introduced into the system by flow injection into a carrier solution -LRB- 100 μL/min -RRB- . During optimization , the carrier solution was water . Injected compounds and ERα , added to the carrier stream via an inverted Y-type mixing union at a flow rate of 100 μL/min , were allowed to bind in a Tefzel reaction coil -LRB- 25 μL -RRB- . This mixture was combined with the flow of the E2-F solution of 100 μL/min . The final equilibrium between ERα , compound , and E2-F was established in a knitted 0.5 mm i.d. PTFE reaction coil -LRB- 250 μL -RRB- . The temperatures of both reaction coils were maintained constant at 37 °C . Fluorescence polarization -LRB- FP -RRB- detection took place directly after the second reaction coil . When the binding of E2-F is decreased due to competition from an eluting ERα ligand , depolarization occurs . This decrease in fluorescence polarization is a measure of the affinity of the ligand towards ERα . Fig. 2Schematic overview of the ERα fluorescence polarization receptor affinity detection -LRB- RAD -RRB- system in high-resolution screening -LRB- HRS -RRB- mode . Samples are injected with an autoinjector -LRB- A.I. -RRB- and are separated using a gradient reversed-phase high-performance liquid chromatography -LRB- HPLC -RRB- system controlled by P1 and P2 , HPLC gradient pumps . After the column the flow is split by a flow spitter , S -LRB- split ratio 1:9 -RRB- . HPLC elution is monitored by UV or fluorescence detection . A makeup gradient , controlled by makeup gradient pumps P3 and P4 , is introduced and eluting compounds are mixed in the first reaction coil with ERα delivered by superloop-1 -LRB- SL-1 -RRB- . In the second reaction coil , the probe ligand E2-F is added to the mix by superloop-2 -LRB- SL-2 -RRB- . ERα affinity detection is performed using a fluorescence detector -LRB- FLD -RRB- . In FIA mode the samples are delivered directly by a carrier solution into the bioassay FP-based ERα receptor affinity detection coupled on-line to gradient HPLC Coupling of the homogeneous ERα FP RAD assay to HPLC was performed in a similar way to that described recently by Kool et al. -LSB- 14 -RSB- for a Cyt P450 affinity assay . When operating the ERα FP RAD system in HPLC mode -LRB- Fig. 2 -RRB- , two pumps were used to control the HPLC gradient and , directly after the HPLC column , the HPLC eluent was split into a 1:9 ratio where nine-tenths of the flow was directed to either an UV -LRB- Agilent 1100 series -RRB- or fluorescence detector -LRB- Agilent 1100 series -RRB- . The choice of detector depended on the compounds to be analyzed . To compensate for increasing the organic modifier concentrations before the delivery of the HPLC eluent to the ERα FP RAD system , two pumps were used to supply a makeup flow consisting of an opposite H2O/MeOH gradient compared with the HPLC gradient in order to keep the MeOH concentration in the assay constant at 10 % . One-tenth of the total makeup flow was combined with one-tenth of the HPLC eluent and this flow was directed to the ERα FP RAD at 160 μl/min . The remaining part of the flow in the ERα FP RAD was the same as described above , with the exception that the HPLC flow combined with the makeup flow replaced the carrier solution flow . For HPLC analysis , test compounds were dissolved in 50 % MeOH in water . For coumestrol , a linear gradient from 75 % MeOH to 99 % MeOH in 7.5 min , constant for 47.5 min , and back to 75 % MeOH in 1 min was applied . The total flow of the HPLC pumps was 162 μL/min and detection took place by measuring UV at 254 nm . For the mixture containing estriol , coumestrol , coumarol , E2 and zearalenone , constant elution at 55 % MeOH for 40 min , followed by a linear gradient from 55 % MeOH to 70 % MeOH in 50 min , constant for 50 min , and back to 55 % MeOH in 1 min was applied . The total flow rate of the HPLC pumps was 229 μL/min and detection took place using UV at 254 nm . For zearalenone and its cytochrome P450-generated metabolites , a linear gradient from 40 % MeOH to 99 % MeOH in 40 min , constant for 30 min , and back to 40 % MeOH in 1 min was applied . The total flow rate of the HPLC pumps was 162 μL/min and detection took place using a fluorescence detector -LRB- λex 279 nm ; λem 440 nm -RRB- . All HPLC separations were carried out on a 150 × 4.6 mm i.d. Luna C18 -LRB- 2 -RRB- column protected with a 2.0 × 5.0 mm i.d. C18 guard column -LRB- Phenomenex , Amstelveen , The Netherlands -RRB- . The HPLC column was thermostated at 25 °C . Results and discussion The aim of this research was to combine a recently developed on-line FP detector cell -LRB- Kool et al. -LSB- 20 -RSB- -RRB- with high-resolution screening -LRB- HRS -RRB- technology to develop a sensitive screening platform for measuring the ERα affinities of individual components in mixtures of compounds without incurring interference from autofluorescence at wavelengths below 450 nm . We first synthesized a suitable tracer compound and used this tracer to set up a FP-based ERα binding assay in FP microplate reader format . The optimized assay conditions for the FP microplate reader format were transferred to an on-line bioassay format in FIA mode . In this mode we subsequently optimized the on-line assay conditions and validated them by measuring IC50 values of known estrogenic compounds in both microplate reader format and in FIA mode . We used the optimized conditions in FIA mode to couple the ERα bioassay on-line to a gradient HPLC system . In HPLC mode we measured the ERα bioaffinities of different estrogenic compounds and screened mixtures with five known estrogenic compounds . Finally , we measured the individual ERα bioaffinities of zearalenone and its metabolites generated off-line by Cyt P450 in HPLC mode . Synthesis of E2-F Synthesis of the fluorescein-labeled derivative E2-F -LRB- Fig. 1 -RRB- was relatively straightforward . The synthetic route described in this paper makes use of readily available chemicals . The yields of intermediate products are relatively high except for the product of the second reaction step , in which compound 2 is converted into 3 . However , the required reactants for this step are not expensive , which makes it possible to perform this step on a large scale . The last and most expensive step involving the coupling of 5-FITC to 7 still gives a yield of 75 % after extraction and purification . Overall , we conclude that this synthetic protocol offers a good alternative to the synthetic scheme described in the literature -LSB- 7 , 19 -RSB- that is used to produce a fluorescein-labeled E2 derivative . We describe a seven-step synthesis with an overall yield of 12 % compared with an eight-step synthesis in the literature with a total yield of 7 % -LSB- 19 -RSB- . To test whether the newly synthesized E2-F had affinity for the ERα , a competitive binding curve for E2-F and ERα LBD was constructed -LRB- data not shown -RRB- using the coumestrol-based microplate reader assay described by Gurer-Orhan et al. -LSB- 6 -RSB- . E2-F was able to displace coumestrol from the ERα binding site , resulting in an IC50 value of 28.2 nM . This value is consistent with KD values found for similar tracers , which were also in the nanomolar range -LSB- 7 , 19 -RSB- . Therefore , we concluded that the synthesized E2-F had affinity for the ERα and could be used to further develop a FP-based microplate reader assay . FP-based microplate reader assay of ERα affinity The first step in the optimization of the FP-based microplate reader ERα affinity assay was the investigation of the influence of E2-F tracer concentration upon the FP-based microplate reader assay response of ERα-ligands . To achieve this we compared the FP signal obtained with 0 % ligand displacement -LRB- no E2 present in the incubation -RRB- with the FP signal with 100 % ligand displacement -LRB- 33.3 μM E2 present in the incubation -RRB- . We also incubated different concentrations of E2-F tracer with a fixed concentration of ERα . The difference in fluorescence polarization -LSB- ΔFP in millipolarization -LRB- mP -RRB- -RSB- was plotted against decreasing E2-F concentrations -LRB- Fig. 3 -RRB- . From this figure it can be seen that ΔFP increases with decreasing tracer E2-F concentrations until it reaches a maximum at 166 nM . However , it should also be noted that when the E2-F tracer concentration decreases , the error bars increase , which means that the S/N ratios become worse . For the ERα-binding assay , we preferred to use a tracer concentration which is not too high , because high tracer concentrations make it necessary to use high concentrations of test compounds as well in order to displace the tracer from the receptor , which results in a decreased assay sensitivity . However , if we use a tracer concentration which is too low , this will also result in unfavorable S/N ratios . Based upon our results and the KD values of 10.4 and 3 nM found by Ohno et al. -LSB- 19 -RSB- and Parket et al. -LSB- 7 -RSB- respectively for structurally similar fluorescein-labeled E2 derivatives , we decided to use an E2-F tracer concentration of 83.3 nM for further optimization experiments . At this concentration the ERα response was high , which makes it easier to detect the tracer -- receptor complex in an on-line assay , with a low S/N ratio , and with the concentration in the same range as the KD value , which makes it possible to determine IC50 values more accurately . Fig. 3FP-based microplate reader tracer concentration optimization . Incubations in the presence of ERα ligand E2 -LRB- 33.3 μM ; equal to 100 % tracer displacement -RRB- were compared with incubations without E2 -LRB- equal to 0 % tracer displacement -RRB- at decreasing E2-F tracer concentrations The second step in the optimization process was the investigation of the influence of the detergent Tween 20 and the solvents methanol -LRB- MeOH -RRB- , acetonitrile -LRB- ACN -RRB- and isopropanol -LRB- iPrOH -RRB- on assay performance using the conditions as optimized in the previous step . Experiments revealed that Tween 20 was not compatible with the ERα FP assay -LRB- data not shown -RRB- . At very low concentrations -LRB- 0.04 % -RRB- , the presence of Tween 20 in the assay caused a large decrease in ΔFP between E2-F bound to ERα -LRB- 0 % displacement ; absence of E2 -RRB- and E2-F displaced by 33.3 μM E2 -LRB- 100 % displacement -RRB- . Although the addition of Tween 20 can give improved resolution in terms of peak width -LSB- 25 -RSB- , we decided not to use it due to the large decrease in the difference in polarization . In Fig. 4 , the effect of increasing concentrations of MeOH , ACN and iPrOH on ΔFP is shown . It can be seen from this figure that ΔFP decreases with increasing organic modifier concentrations . ΔFP decreased to almost zero for ACN at the highest concentration . For MeOH and iPrOH , however , a signal difference between the E2-F bound to ERα and displaced by E2 could still be observed at the highest modifier concentration . Oosterkamp et al. -LSB- 10 -RSB- have stated that denaturation of the estrogen receptor occurs upon prolonged contact with organic solvents such as MeOH and ACN . It is therefore reasonable to assume that when the organic modifier is present at higher concentrations , the denaturation process for ERα LBD increases , which results in smaller fluorescence polarization signal differences . Fig. 4FP-based microplate reader solvent optimization . Incubations in the presence of ERα ligand E2 -LRB- 33.3 μM ; equal to 100 % tracer displacement -RRB- were compared with incubations without E2 -LRB- equal to 0 % displacement -RRB- at increasing MeOH , ACN and iPrOH concentrations at an E2-F concentration of 83.3 nM FP-based ERα receptor affinity detection -LRB- RAD -RRB- in flow-injection analysis -LRB- FIA -RRB- mode The optimized microplate reader assay conditions were transferred to the on-line ERα RAD system in FIA mode -LRB- Fig. 2 -RRB- . In this system , compounds introduced by a carrier solution are allowed to bind to the ERα LBD in a first reaction coil , and the E2-F tracer is introduced after this coil . The tracer compound can interact with the unbound receptor in a second reaction coil and detection takes place after the second reaction coil using FP detection . In a FIA setup , carrier solution replaces the gradient HPLC system , thereby allowing the direct injection of compounds and the optimization of on-line assay conditions before final on-line coupling to HPLC . The influence of the ERα LBD and E2-F tracer concentrations and the corresponding excitation and emission wavelengths were investigated in FIA mode in order to optimize the performance of the FP ERα RAD system . The influences of ACN and MeOH , later used in HPLC mode too , on the FP ERα RAD system performance were also tested in the FIA mode . All measurements were performed in duplicate . ERα LBD and E2-F tracer optimization Different receptor concentrations -LRB- ERα LBD -RRB- were tested in combination with different tracer concentrations -LRB- E2-F -RRB- by varying the flows of SL-1 -LRB- containing 10 nM ERα in binding buffer -RRB- and SL-2 -LRB- containing 1 μM E2-F in binding buffer -RRB- . The total flow rate was kept constant by introducing a compensating superloop containing only binding buffer . Water was used as carrier solution in order to inject 10 μL 100 μM E2 in 50 % MeOH samples -LRB- 100 % ligand displacement -RRB- in duplicate followed by 10 μL injections of 50 % MeOH -LRB- 0 % displacement -RRB- at a flow rate of 250 μL/min . The results obtained with these experiments are depicted in Fig. 5 . This figure shows that as the E2-F tracer concentration increases the FP signal increases too , until it reaches a maximum . Using the present FIA FP ERα RAD setup , we were able to determine the KD and maximum response -LRB- Rmax -RRB- values for the E2-F tracer compound -LRB- Table 1 -RRB- at different ERα concentrations . The KD values found were of the same order as observed by Ohno et al. -LSB- 19 -RSB- and Parker et al. -LSB- 7 -RSB- for an analogous FP tracer compound . It can be seen that Rmax increases linearly with higher ERα concentrations , as was expected . Based upon these results , we conclude that the FP ERα RAD in FIA mode worked well , and we continued by investigating the influence of the organic modifier upon the assay performance . Fig. 5Determination of KD and maximum response -LRB- Rmax -RRB- for E2-F in FIA mode . Different concentrations of E2-F were incubated with three different ERα concentrationsTable 1KD and maximum response -LRB- Rmax -RRB- determination for E2-F in FIA mode -LSB- ERα -RSB- -LRB- nM -RRB- KD -LRB- nM -RRB- Rmax -LRB- AUC -RRB- a0 .743.6 ± 12.42.17 × 1061.526.4 ± 5.04.04 × 1063.034.5 ± 5.39.38 × 106aRmax was determined by integrating the decrease in FP signal , and arbitrary units were chosen to display this decrease Organic modifier concentration Although we also investigated the influence of iPrOH upon the ERα bioaffinity assay performance in the FP-based microplate reader format , we decided to evaluate only MeOH and ACN in FIA mode based upon the fact that we planned to use only these two solvents in HPLC mode . Different concentrations of MeOH and ACN were used as the carrier solution in order to inject 10 μL 100 μM E2 ligand in 50 % MeOH samples -LRB- 100 % ligand displacement -RRB- in duplicate followed by 10 μL injections of 50 % MeOH -LRB- 0 μM E2 ligand ; 0 % displacement -RRB- into the system . The ERα LBD concentration in the second reaction coil was 2.7 nM , the E2-F tracer concentration 110 nM and the total flow rate 300 μL/min . Increasing the concentration of MeOH decreased the S/N ratio in FIA mode -LRB- Fig. 6 -RRB- . A similar effect was observed with ACN . A possible explanation for this decreasing effect is the partial denaturation of the estrogen receptor due to the presence of high concentrations of organic modifier . Oosterkamp et al. -LSB- 10 -RSB- have described this phenomenon during the optimization of the ERα coumestrol-based RAD system when a decrease in response was observed upon the contact of the ERα with organic solvents such as MeOH and ACN . One advantage of a higher organic modifier concentration is that higher flow rates can be used in HPLC gradients , which may result in shorter analysis times and higher resolutions . Another advantage is that high concentrations of organic modifier prevent lipohilic compounds from adsorbing to the walls of the reaction coils and other tubing , which may increase resolution . Based upon our results during FP FIA setup , we concluded that both MeOH and ACN might be used in the on-line gradient HPLC system . Fig. 6Effect 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 When the excitation and emission wavelengths were varied , the optimal FP signal responses in FIA mode were obtained at an excitation wavelength of 498 nm and an emission wavelength of 522 nm -LRB- data not shown -RRB- . These wavelengths were therefore used to further optimize the on-line FP system in FIA mode . Competitive receptor binding curves To validate the ERα FP RAD system , competitive binding curves for five known estrogenic test compounds -- DES , E2 , EE2 , tamoxifen and coumestrol -- were constructed using the FP-based microplate reader assay , and these values were compared with the IC50 values obtained using the ERα FP setup in FIA mode . The ERα LBD concentration was 1.6 nM and the E2-F tracer concentration was 80 nM for the microplate reader assay . In FIA mode , the ERα LBD concentration in the second reaction coil was 2.7 nM and the E2-F concentration was 110 nM . All five test compounds displayed sigmoidal concentration -- response curves during both the microplate reader setup and in FIA mode , and from these curves we were able to determine the IC50 values -LRB- Table 2 -RRB- . The values found in the two different setups were similar for each individual compound and in agreement with values obtained from the literature -LSB- 7 , 26 -RSB- . We thus demonstrated that the FP-based microplate reader assay and the ERα FP setup in FIA mode could be employed to determine the IC50 values of a wide range of typical ERα-binding ligands accurately . The next step in the process of developing the HRS platform was to couple the FIA ERα FP system to a gradient HPLC system on-line . Table 2IC50 values determined in FIA mode and in microplate reader assay formatCompoundFIA mode IC50 -LRB- M ± SEM ; n = 3 -RRB- Microplate setup IC50 -LRB- M ± SEM ; n = 3 -RRB- Literature data IC50 -LRB- M -RRB- DES6 .9 ± 0.3 × 10 − 96.1 ± 0.6 × 10 − 93.5 × 10 − 9 aTamoxifen1 .2 ± 0.1 × 10 − 71.3 ± 0.4 × 10 − 71.9 × 10 − 7 aCoumestrol1 .8 ± 0.2 × 10 − 72.5 ± 0.3 × 10 − 73.6 × 10 − 7 bE26 .7 ± 0.8 × 10 − 93.3 ± 0.3 × 10 − 95.6 × 10 − 9 aEE21 .6 ± 0.2 × 10 − 93.5 ± 0.8 × 10 − 92.3 × 10 − 9 bIC50 values of five estrogenic compounds towards the ERα , as measured with the RAD system in FIA mode and with the microplate reader assay format . Data from literature a -LSB- 7 -RSB- , b -LSB- 26 -RSB- are also shown . DES , diethylstilbestrol ; E2 , 17β-estradiol ; EE2 , 17α-ethinyl estradiol FP-based ERα receptor affinity detection coupled on-line to HPLC Following assay optimization in the microplate reader and FIA modes , the ERα FP RAD assay was coupled on-line to gradient HPLC . In this HRS setup the flow eluting from an HPLC column was coupled directly to the FP RAD system , which enables compound separation prior to the screening of individually eluting compounds for their ERα affinities . This allowed us to screen for compounds with ERα affinities in mixtures . To test the performance of the on-line HRS FP ERα system , different concentrations of the phytoestrogen coumestrol were injected first . A negative peak in the ERα bioaffinity trace was observed at a retention time of 40 min , which is the exact time when coumestrol eluted from the HPLC column -LRB- data not shown -RRB- . As expected , the negative bioaffinity peaks became larger upon injections of higher concentrations of coumestrol . The highest negative peaks were observed when all of the E2-F was displaced by coumestrol . Higher concentrations of coumestrol only caused more tailing of the bioaffinity peaks . These measurements illustrate that the ERα bioaffinity of the phytoestrogen coumestrol could be measured sensitively and efficiently with the new E2-F based HRS ERα FP system , which is not possible due to the occurrence of autofluorescence using the coumestrol-based ERα HRS system developed by Oosterkamp et al. -LSB- 10 -RSB- . Representative superimposed chromatograms of different concentrations of a mixture of five known estrogenic compounds -- coumestrol , coumarol , estriol , 17β-estradiol and zearalenone -- are depicted in Fig. 7 . Although some baseline drift in time is observed over two hours , it is quite possible to detect the ERα bioaffinities of all five individual peaks in the mixture . The explanation for the baseline drift is most likely that the post-column makeup HPLC gradient does not totally compensate for the effect of variations in the organic modifier concentration . The FP ERα RAD system is sensitive to changes in the organic modifier concentration , as shown during FIA optimization , and this can lead to baseline drifts . Fig. 7ERα FP RAD trace of a mixture of five estrogenic compounds injected into the ERα FP RAD HPLC system -LRB- injected compounds are as follows : estriol -LRB- 1 -RRB- -LSB- 3.75 nmol ; 49.5 min -RSB- , coumestrol -LRB- 2 -RRB- -LSB- 3.75 nmol ; 53.7 min -RSB- , coumarol -LRB- 3 -RRB- -LSB- 37.5 nmol ; 80.4 min -RSB- , 17β-estradiol -LRB- 4 -RRB- -LSB- 3.75 nmol ; 102.8 min -RSB- and zearalenone -LRB- 5 -RRB- -LSB- 12.5 nmol ; 112.5 min -RSB- To illustrate another application of the on-line HRS ERα FP system , metabolites of the phytoestrogen zearalenone , which were anticipated to show affinity for the ERα , were analyzed in the present FP-based HRS ERα system . For this purpose , zearalenone was metabolized by phenobarbital -LRB- PB -RRB- - induced rat liver microsomes in an off-line fashion , followed by analysis with the FP-based HRS system . The results are depicted in Fig. 8 . The UV trace of the t = 0 min incubation shows that zearalenone -LRB- Z -RRB- elutes at 51.4 min . The corresponding bioaffinity trace shows that zearalenone has affinity for ERα . The FLD trace of the t = 120 min incubation shows that zearalenone is metabolized by rat liver microsomes into at least three major metabolites -LRB- M1 : 33.5 , M2 : 39.5 and M3 : 43.2 min -RRB- . The corresponding bioaffinity trace demonstrates that all three major metabolites -LRB- M1 -- 3 -RRB- have affinity for ERα . LC/MS measurements were performed to identify the three major metabolites . M1 had a molecular ion at an m/z value of 333 , which likely corresponds to a monohydroxylated zearalenone metabolite . M2 had a molecular ion at an m/z value of 317 , which is the exact same m/z value as that of zearalenone itself . This can be explained by the relocation of the double carbon -- carbon bond next to the aromatic ring . However , more research must be performed to confirm this . M3 had a molecular ion at an m/z value of 319 , which corresponds well to the m/z value of the zearalenone metabolite zearalenol described in the literature -LSB- 27 -RSB- . The metabolism of zearalenone in vitro by rat liver microsomes has been described very recently by Pfeiffer et al. -LSB- 28 -RSB- , and they confirm the findings of M1 and M3 . The present Cyt P450 metabolism experiment illustrates that it is possible to screen metabolic mixtures of phytoestrogenic compounds for ERα affinity using the present FP-based HRS ERα setup . However , no further structural elucidation of the zearalenone metabolites has been performed , because the goal of the present experiment was only to show that the on-line FP HRS ERα assay can be used to screen for parent compounds and metabolites of phytoestrogens with ERα affinity . Fig. 8a -- ba HPLC FLD chromatograms of zearalenone -LRB- Z -RRB- incubated for 0 and 120 min with PB-induced rat liver microsomes . M1 , M2 and M3 are metabolites formed during the 120-min incubation period . b The corresponding ERα FP affinity traces . The structure of zearalenone is depicted in the figure We conclude that we succeeded in developing , for the first time , an on-line FP-based HRS platform to screen for ERα affinity using the novel on-line FP detector cell recently described in a FIA mode by Kool et al. -LSB- 20 -RSB- . The sensitivity of the FP-based HRS platform is comparable to that of the coumestrol-based HRS platform , and no interference from fluorescence was observed at wavelengths below 450 nm . The novel on-line FP detector cell can in principle be applied to other HPLC-based bioaffinity assays which require FP detection . The present HRS ERα FP system offers a novel technology for investigating the metabolic profiles of drugs and other compounds such as phytoestrogens which can not be measured by the coumestrol-based HRS system due to the occurrence of autofluorescence from the test compounds .

O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O

fluorescence polarization
estrogen receptor α
phytoestrogens

high-resolution screening (hrs)
on-line bioaffinity assay
receptor affinity detection (rad)