Quantification of circulating steroids in individual zebrafish using stacking to achieve nanomolar detection limits with capillary electrophoresis and UV-visible absorbance detection.

Capillary electrophoresis and UV-visible absorbance detection are used with sample stacking to achieve detection limits ranging from 0.2 to 2 ng/mL (0.8 to 6 nM) for steroids. Stacking is accomplished using negatively charged cyclodextrin steroid-carrier molecules at a discrete pH interface between the reconstituted sample and the separation electrolyte. Steroids are then separated in under 5 min using capillary electrophoresis that incorporates secondary equilibria via sodium dodecyl sulfate and cyclodextrin. The effectiveness of the method for measurements of multiple steroids in limited sample volumes is demonstrated in individual female fish with total circulating blood volumes of 5 µL or less. Steroid recoveries from plasma following a sample processing method developed with commercial extraction cartridges range from 81 to 109 % for 17α,20ß-dihydroxy-pregn-4-en-3-one, testosterone, 11-ketotestosterone, estrone, 17ß-estradiol, and 17α-ethinyl estradiol. When applied to reproductively active female zebrafish, changes were detected in the levels of circulating steroids as a result of exposure to different solvents and 17ß-estradiol.

Assessment of aptamer-steroid binding using stacking-enhanced capillary electrophoresis.

The binding affinity of 17ß-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17ß-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 µM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.