Use of Candida albicans and its oestrogen binding protein as a bio-recognition element for detection of oestrogen

Abstract

Compared with bacteria, yeast have rarely been studied for use as a biocomponent for biosensors. Yeast are easy to culture and are eukaryotes, which means their biochemistry in many respects is similar to that of higher organisms. C. albicans and some other yeast species are known to have an oestrogen binding protein (EBP), which oxidises NAD(P)H to NAD(P)+ Oestrogen, when present, binds to the NAD(P)H oxidation site which leads to an accumulation of NAD(P)H (Madani, et al. 1994). Previous research has shown that oestrogen can be quantified using S. cerevisiae whole cells as the detection element and measuring NAD(P)H with a double mediator electrochemical system. This thesis employs the mediated electrochemical systems to investigate the influence of growth phase on EBP production in C. albicans and the response of cells of different ages to different concentrations of oestrogen. A cell not known to possess EBP (Arxula adeninivorans) was also investigated for its response to 17β-oestradiol. As expected, A. adeninivorans did not show a detectable response to 17β-oestradiol but surprisingly, its catabolism was inhibited. By using C. albicans cell lysate in the oestrogen detecting assay, utility was systematically increased and the complexity of the whole cell assay was decreased. In this assay, only a hydrophilic mediator was used, removing the need for a lipophilic mediator. The assay was used successfully in a complex medium, the upper detection limit was raised to 100 nM of 17β-oestradiol, and the assay period was reduced to 20 min. The electrodes were modified to directly detect NAD(P)H in cell lysate at a lower potential to avoid interference by oxidants such as ascorbic acid. Furthermore, EBP was purified using 17β-oestradiol affinity chromatography, and the protein was used with NADH in an oestrogen bioassay. In this assay NADH was electrochemically detected directly and could differentiate ‘with’ and ‘without oestrogen’ samples. This research also showed that a mediator can interact directly with EBP, i.e. without the use of NADH and further, direct electron transfer from EBP to both glassy carbon and pyrolyzed photoresist film (PPF) electrodes were demonstrated (i.e. without the use of a mediator). This research has further simplified the assay and will facilitate the development of rapid oestrogen detecting protein biosensor.