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The widespread use of natural and synthetic estrogens or chemicals with estrogenic activities is causing an increasing accumulation of estrogenic compounds in the environment. Already at very low concentrations these estrogenics can severely affect the wildlife, particularly in an aquatic environment. For these reasons measuring devices for detecting estrogen contaminations are in great demand. The majority of the analytical methods and bioassays on the market so far, lack semi-online adaptability, and usually cannot be used for automatic and continuous determination. Therefore, we have embarked on the development of new systems, which are able to fulfil those demands. The EstraMonitor combines recombinant A. adeninivorans G1212/YRC102-hERa-phyK yeast cells as the microbial component with an amperometric detection method to analyze estrogenic contaminations. A. adeninivorans G1212/YRC102-hERa-phyK was constructed by Kaiser et al. (2010). These cells were engineered to co-express the human estrogen receptor (hERa) gene and the inducible phytase (phyK, derived from Klebsiella sp. ASR1) reporter gene under control of a promoter with estrogen response elements (EREs). In the presence of estrogenic substances, such as 17ß -estradiol (E2), the phyK gene is expressed and recombinant phytase is secreted into the media. The level of phytase is quantified by amperometric detection using substrate p-aminophenyl phosphate (p-APP). Phytase dephosphorylates p-aminophenyl phosphate (p-APP) into an intermediate product p-aminophenol (p-AP). p-AP is electroactive and oxidized at the electrode. This generates electrons and produces a current which is proportional to the level of phytase activity. Since phytase activity is directly correlated to the E2 concentration, the estrogenic activity can thus be calculated from the current measured. The microbial component of the EstraMonitor, the non-immobilized A. adeninivorans G1212/YRC102-hERa-phyK, works well with the amperometric method in a quantitative manner. The optimal applied potential determined for amperometric measurements was 150 mV and provided a low background signal for the amperometric detection. The half maximal effective concentration (EC50) and limit of detection (LoD) values for E2 obtained from amperometric measurements with the EstraMonitor were 69.9 ng L-1 and 44.5 ng L-1, respectively. The measuring procedure of the EstraMonitor system including incubation of A. adeninivorans G1212/YRC102-hERa-phyK cells with E2, subsequently incubation with electrochemical substrate (p-APP), and signal recordation is completed within only 4 h and 10 min. Out of this total time, amperometric detection including substrate incubation and signals recordation takes only 10 min out of total time. The use of immobilized cells for a microbial biosensor is an essential advantage of the EstraMonitor system because it allows easy-handiness next to long-term stability and reusability. Immobilized A. adeninivorans G1212/YRC102-hERa-phyK cells revealed excellent properties which make them very suitable for semi-online, automatic and continuous monitoring. They were stable up to 30 days when stored at 4 °C. Furthermore, they could be reused up to 15 times. The EC50 and LoD values achieved for E2 using immobilized cells in combination with amperometric detection were 20.9 and 8.3 ng L-1, respectively. Furthermore, this application also removes the need to separate cells by centrifugation, to sterilize the samples as well as to cultivate repeatly. Additionally, both immobilized and non-immobilized A. adeninivorans G1212/YRC102-hERa-phyK cells remain fully functional in a wide range of untreated wastewater samples and in environments containing up to 5% NaCl. To enhance the sensitivity and reduce the time for estrogenic determination, an alternative A. adeninivorans G1214/YRC103-hERa-phyK strain was developed. This strain can produce a detectable amount of phytase within 2 h after induction with E2. It offers an improved microbial component in terms of sensitivity and time-effectiveness. In addition, to reduce the cost for estrogenic detection an alternative substrate, ascorbic acid 2-phosphate (AA2P), was tested. AA2P, which is both cheap and widely available, performed better than p-APP. The EC50 and LoD values for E2 obtained with AA2P were 15.69 and 0.92 ng L-1 versus 20.09 and 8.3 ng L-1 when examined with p-APP, respectively. Taken together, the EstraMonitor is an automated system with respect to sample cycling, sample measuring and calibration supplemented with an alarm function. This system makes it possible to control estrogenic activity semi-online, automatically and continuously. These are advantages of the EstraMonitor compared to other estrogenic detection systems. It can thus be concluded that, the EstraMonitor is a powerful and feasible semi-online device for monitoring estrogenic activity especially adapted for the use in sewage treatment plants.
The following work is describing the development of two innovative biosensors for the detection of biologically relevant molecules in the field of ecology and medical diagnostics. Biosensors have the particularity to possess a biological partner which recognizes the target molecule and a physical detection method responsible for the transformation of this biological interaction into measurable information. In the present case, both biosensors are designed following the same strategy and use a recombinant produced human receptor as biological partner and the surface plasmon resonance (SPR) technique to transform the biological interaction in quantitative information. The progesterone biosensor is aimed to detect and quantify substances with affinity to the human progesterone receptor. The recent discoveries that some chemicals present in low quantities in the ecosystem called endocrine disrupting chemicals (EDCs) have a negative impact on the aquatic life fitness raised concerns about the effects of these same molecules to the human health. In order to assess the effects of these EDCs, the use of classical analytical detection methods like high performance liquid chromatography (HPLC) or gas chromatography (GC) is not sufficient as these techniques only quantify a defined molecule without giving information about its biological activity. By integrating a recombinant human progesterone receptor, the progesterone biosensor can determine the biological activity of an unknown molecule or of a mixture of molecules in a real sample. In this work, two different yeasts – one methylotrophic (Hansenula polymorpha) and one non-methylotrophic (Arxula adeninivorans) - were selected as host for the recombinant protein production and their performances were compared. Different purification strategies were assayed and the binding activity of the purified progesterone receptor was then confirmed by enzyme like receptor assay (ELRA) and SPR. This led to the design of a first version of the biosensor with the immobilization of a progesterone-BSA ligand to the surface of a SPR chip and the use of a progesterone receptor mixed with the target molecule as sample. This competitive assay format was successfully utilized with a commercial progesterone-BSA ligand as target molecule and the next step will be the adaptation of this biosensor for real samples measurements. The HER-2 biosensor was developed as an answer for one of the most critical issue in the field of breast cancer diagnostics. In approximately 30 % of cancer cases, the transmembrane protein HER-2 can be found in large amount at the surface of the carcinoma cells and these cases are known to be particularly aggressive. Based on the amount of HER-2 protein at the surface of the cells, the pathologists established a scale with four levels to adapt the treatment to each patient. Although effective therapies have been developed to treat the HER-2 positive breast cancer, one of the major challenges remains the classification of breast sample in this scale as the only accepted determination methods are immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) which are only qualitative. In this work, a biosensor has been designed to quantify the amount of the HER-2 protein in a crude cell extract from a breast cancer tissue sample. To achieve this, the strategy is to utilize an antibody specifically targeted against the HER-2 protein and bound to a SPR chip. As the development of this biosensor necessitated the use of large amount of purified HER-2 protein, it was decided to produce recombinant full-length HER-2 in two different yeasts and to purify it by chromatography. This recombinant protein production required particular attention due to the membrane localization of HER-2. The structural integrity of the recombinant protein was confirmed by Western Blot and ELISA and different antibodies were bound to SPR chips in order to detect the HER-2 protein. After finding the conditions giving an optimal SPR signal, a protocol was developed to extract native HER-2 from breast tissue sample and the biosensor was assayed with this crude cell extract.