TECHNISCHE UNIVERSITÄT MÜNCHEN Development of Immunological Methods for the Detection of Micropollutants in Fresh Water Sample
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TECHNISCHE UNIVERSITÄT MÜNCHEN Institut für Wasserchemie und Chemische Balneologie Lehrstuhl für Analytische Chemie Development of Immunological Methods for the Detection of Micropollutants in Fresh Water Samples Maria Hübner Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grads eines Doktors der Naturwissenschaft genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. Michael Groll Prüfer der Dissertation: 1. Univ.- Prof. Dr. Reinhard Nießner 2. apl. Prof. Dr. Dietmar Knopp Die Dissertation wurde am 04.09.2015 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 18.11.2015 angenommen. Acknowledgements This thesis was prepared during August 2012 and July 2015 at the Chair for Analytical Chemsitry of the TU München under supervision of Univ-Prof. Dr. Reinhard Nießner. The work was financially supported by the DFG and ANR in the project “Nanoscaled architectures for highly sensitive biosensing of small molecules”. I want to address a special thank to Prof. Dr. Reinhard Nießner for the friendly reception at the institute and for giving me the trust necessary to accomplish my thesis. Further, I want to thank Prof. Dr. Dietmar Knopp for his academic supervision and faithful guidance throughout my work. I appreciate his kind and patient way in leading the Bioanalytics group. Thank you for always taking time for discussion. Warmly, I want to thank Prof. Dr. Souhir Boujday from the University Pierre et Marie Curie in Paris (France). Her input made this work more versatile in an academic and personal way, bacause I could spend several weeks in 2013 and 2014 in her laboratory in Ivry-sur-Seine. I apreciate her efforts to supervise me and my experiences in Paris very much. I thank Joachim Langer for the analysis of water samples, Helmut Krause for the measurements of MALDI-TOF mass spectra and Christophe Méthivier for XPS measurements. I adress special thanks to Susanna Mahler for the support with LC-FLD measurements and the friendly atmosphere in the laboratory. My fellow student Maroua Ben Haddada, and Master student Verena Fink, as well as the Bachelor students Jan Berger and Alexander Urstöger for their high interest in the project and their help in the laboratory. For proof-reading this thesis, I want to thank Verena Meyer, Maroua Ben Haddada and Prof. Dr. Souhir Boujday. I thank all my colleagues at the IWC for their constant helpfulness. A special thanks to my collaborates in the Bioanalytic group: Anna-Catherine Neumann, Xu Wang, Dr. Susanna Oswald and Dr. Michael Pschenitza. I want to express my gratefulness towards my dear collegues Verena Meyer and Anika Wunderlich for their good mood and support during my time at the institue. At last but not at least, I want to thank my family for their absolute belief in me and my skills. Publications Parts of this thesis have been published in the following scientific journals: Huebner M., Weber E., Boujday S., Niessner R., Knopp D., Rapid analysis of diclofenac in fresh water and wastewater by a monoclonal antibody-based highly sensitive ELISA. Anal. Bioanal. Chem . 2015 , 407 , 8873-8882. Huebner M., Ben Haddada M., Méthivier C., Niessner R., Knopp D., Boujday S., Layer-by- layer generation of PEG-based regenerable immunosensing surfaces for small-sized analytes. Biosens. Bioelectron. 2015 , 67 , 334-341. Mercier D., Ben Haddada M., Huebner M., Knopp D., Niessner R., Salmain M., Proust A., Boujday S., Polyoxometalate nanostructured gold surfaces for sensitive biosensing of benzo[a]pyrene. Sens. Actuators B, 2015 , 209, 770-774. "Inmitten der Schwierigkeiten liegt die Möglichkeit." Albert Einstein Contents I Introduction .................................................................................................................... 1 II Fundamentals ................................................................................................................ 4 1 Micropollutants in fresh water ............................................................................................ 5 1.1 Benzo[a]pyrene as an indicator for pollution by Polycyclic Aromatic Hydrocarbons (PAHs) ................................................................................................................................. 7 1.2 Diclofenac as an example for pollution by pharmaceuticals ..................................... 10 1.3 Microcystin-LR: a cyanobacteria-born toxin .............................................................. 12 2 Immunoassays for the analysis of water samples ............................................................. 16 2.1 Antibodies .................................................................................................................. 16 2.2 Preparation of monoclonal and polyclonal antibodies .............................................. 18 2.3 Characterization of monoclonal antibodies ............................................................... 20 2.4 Competitive ELISA ...................................................................................................... 21 2.5 Evaluation of immunoassays ...................................................................................... 23 2.6 ELISAs for fresh water monitoring applications ......................................................... 25 3 Biosensors and microarrays for the analysis of fresh water samples ............................... 26 3.1 Overview on existing biosensors ................................................................................ 26 3.2 Setup for microarray-based micropollutant detection .............................................. 28 3.3 Preparation of regenerable immunosensors on glass-type surfaces ........................ 30 3.4 Methods for surface chemistry analysis during biosensors preparation .................. 32 3.4.1 Atomic force microscopy .................................................................................... 33 3.4.2 Water contact angle ............................................................................................ 33 3.4.3 Grazing-angle attenuated total reflection FT-IR ................................................. 35 3.4.4 X-ray photoelectron spectroscopy ...................................................................... 36 3.4.5 Quartz crystal microbalance with dissipation measurement ............................. 37 4 Chemiluminesence as a detection principle for biosensors and immunoassays ............... 39 Contents III Results and Discussion ................................................................................................ 40 1 Development of monoclonal anti-DCF antibodies for fresh water analysis ...................... 41 1.1 Identification of monoclonal anti-DCF antibodies with high affinity by screening hybridoma cell culture supernatants ............................................................................... 41 1.2 Characterization of purified monoclonal antibodies ................................................. 43 1.2.1 MALDI-TOF-MS spectra ....................................................................................... 43 1.2.2 SPR sensor surface design and binding curves ................................................... 45 1.2.3 Cross reactivity .................................................................................................... 50 1.3 Effect of organic solvents on antibody affinity in ELISA ............................................. 53 1.4 Protocol optimization ................................................................................................. 54 1.5 Matrix effects in fresh water ...................................................................................... 57 1.6 Analysis of fresh water ............................................................................................... 62 1.7 Analysis of wastewater .............................................................................................. 63 2 Surface chemistry aspects for sensitive immunosensing of small analytes ...................... 66 2.1 Stability and reversibility of antibody binding to haptens on PEG modified surface preparations in QCM-D experiments ............................................................................... 66 2.2 Microspotting of small molecules .............................................................................. 69 2.2.1 A microspotting method for the immobilization of small molecules ................. 70 2.2.2 Optimization of the microspotting method and calibration of the flow-through ELISA for DCF ................................................................................................................ 74 2.2.3 Development of a microspotting method for MC-LR ......................................... 77 2.2.4 Optimization of the microspotting method and calibration procedure for the detection of BaP ........................................................................................................... 79 2.3 Surface characterization of a regenerable surface preparation ................................ 85 2.3.1 AFM ..................................................................................................................... 85 2.3.2 Contact angle measurement ............................................................................... 87 2.3.3 XPS ....................................................................................................................... 87 2.3.4 GA-ATR FT-IR ......................................................................................................