Development, characterization of organic solvent resistant bacteria based microbial biosensor and molecularly imprinted polymer based biosensor systems and use in determination of some steroids

The determination of steroids is generally performed by immunoassay methods and chromatographic methods. Such methods include time consuming preprocessing and expensive equipment. However, using microbial biosensors and MIP based electrochemical sensors, rapid, sensitive, inexpensive and selective steroid detection can be performed. In this thesis, MIP sensors for cortisol-cortisone and prednisolone and microbial biosensor system for cholesterol have been developed. MIP sensors were prepared by electropolymerization method in the presence of pyrole and template molecule on gold nanoparticle coated graphite electrode surface. Sensor responses were monitored by Ferri / Ferro solution with Differential Pulse (DP) method. Template molecule / monomer ratio, incubation and extraction duration optimizations were performed in the MIP sensors that developed for cortisone-cortisol and prednisolone. The linear detection range of the Cortisone-MIP sensor was found to be 1-100 M and the detection limit 0.25 M, while the linear detection range of the Prednisolon-MIP sensor was found to be 1-75 M and the detection limit 0.35 M. In the reproducubility experiments average value (¯x), standard deviation (±) and variation coefficient (CV %) values for the 50 M cortisone (n = 10) by the Cortisone-MIP sensor were calculated to be 48.10 M, ± 2.42 M and 5.04%, respectively, and for the prednisolone (n = 10) were found as 48.69 M, ± 1.06 M and 2.189% respectively. MIP sensors have been used to test substrate specificity, interference effect, and storage stability. In addition, the developed MIP sensors were tested for cortisol-cortisone and prednisolone in lyophilized urine and serum samples. Within the microbial biosensor development step, cholesterol metabolism induced and organic phase tolerant Pseudomonas sp. and catalase enzyme were used as a biocomponent of biosensor. Pseudomonas sp. and the catalase enzyme were immobilized into multi-wall carbon nanotube (MWCNT) / ionic liquid (IL) / carbon paste electrode (CPE). Immobilization steps of the biosensor were characterized by CV and impedance spectroscopy. Hydroquinone was used as the mediator system in the measurements. The amperometric working potential was found to be -0.2 V. Optimization and characterization studies were also carried out by amperometric method. The optimization of working conditions of microbial biosensor were carried out and optimum temperature and pH were found to be 35 °C and 7.0 (Phosphate buffer, 50 mM). The effect of amounts of bacteria on the biosensor responses was investigated. The linear range of this microbial biosensor that developed for the determination of cholesterol was found to be 5-600 M and the detection limit was found as 1.52 M. The effect of steroid compounds such as cortisone, cortisol, testosterone, β-sitosterol and estradiol, and the presence of endogenous analytes such as glucose and ascorbic acid on the biosensor response was investigated in substrate specificity and interference effect experiments. After optimization and characterization studies of the microbial biosensor, the biosensor response to cholesterol in saturated cyclohegzan was investigated and also responses obtained were compared with response obtained by water phase. Cholesterol determination was carried out both in cyclohegzan and in water phase by developed microbial biosensor.