Electrochemical detection of oestrogen binding protein interaction with oestrogen in Candida albicans cell lysate

Previously we reported an electrochemical method to quantitatively detect vertebrate oestrogens using wild type Saccharomyces cerevisiae cells. That assay required the use of a double mediator system, a five-hour incubation period and had a maximum detection limit of around 11 nM 17β-oestradiol. In the work reported here we have sought to systematically increase the utility and decrease the complexity of the whole cell assay. The steps we took to achieve this goal were in order; lysing the cells to remove transport constraints, removing the lipophilic mediator and conducting the assay with the hydrophilic mediator only and finally performing the assay in a complex medium to demonstrate its specificity. Linear sweep voltammetry was used to investigate the interaction of mediators with NADH. The assay is now cell free and functions in a complex substrate. The linear response range upper limit has been raised to 100 nM with a calculated limit of detection of 0.005 nM with a limit of determination of 0.014 nM and the assay period has been reduced to 20 min.