Characterisation of electrochemical detection of oestrogen by using yeast oestrogen binding protein
Thesis DisciplineBiological Sciences
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
Oestrogens are female sex hormones. Both natural and synthetic oestrogens have been found in many aquatic environments. There are three naturally occurring oestrogens – oestradiol, estriol and estrone. Oestradiol (correctly known as 17β oestradiol or E2) is a naturally occurring steroid hormone and is the most potent of these three. Diethylstilbestrol, dienestrol, quinestrol etc are synthetic oestrogens. These environmental steroidal and nonsteroidal oestrogens act as endocrine disruptors (EDs). Both types of oestrogens in the environmental samples can be quantified using several laboratory methods such as high pressure liquid chromatography (HPLC), gel permeation chromatography (GPC) etc. but they often require extensive training to perform. Arxula adeninivorans is biotechnologically significant dimorphic yeast with unusual characteristics. It can use a wide range of substrates and it is thermotolerant, osmotolerant and halotolerant. It is a non-pathogenic fungus and is therefore ideal for use in industrial settings. It is a source of many enzymes and a wide range of transformation platforms have been developed to enable the production of foreign proteins. In this project, A.adeninivorans was transformed with histidine-tagged synthetic oestrogen binding protein (EBP) gene based on the Candida albicans EBP sequence. The recombinant EBP expressed in the yeast Arxula is separated using HisTrap columns. Linear sweep voltammetry was used for the detection of EBP redox responses to oestrogen in solution. A three-electrode configuration was used for all measurements [auxiliary electrode (platinum wire), reference electrode (Ag/AgCl) and working electrodes (Pt 50μm diameter micro-disc and 2 mm diameter glassy carbon)]. Electron transfer from EBP to electrodes will require the use of a mediator system and TMPD, a lipophilic mediator used in this experiment. Screen printed electrodes (SPEs) were used to detect the interaction between EBP and oestrogen. To perform experiment with SPE, EBP was immobilized on SPE using the crosslinker glutaraldehyde. Differential pulse voltammetry (DPV) was used to detect interactions of EBP and oestrogen on SPE. Immobilion N transfer membrane was impregnated with TMPD solution and electrochemistry (DPV) was performed. The purpose of using membrane is to simulate the immobilization of TMPD on SPE along with EBP for the detection of oestrogen.