Immunoanalytical Determination of Mycotoxins in Food with an Automatized Instrumental Platform [Elektronische Ressource] / Jimena Celia Sauceda-Friebe. Gutachter: Reinhard Niessner ; Peter Schieberle. Betreuer: Reinhard Niessner

Jimena Celia

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Institut für Wasserchemie und Chemische Balneologie
Lehrstuhl für Analytische Chemie
Immunoanalytical Determination of Mycotoxins in Food with an
Automatized Instrumental Platform
Jimena Celia Sauceda-Friebe
Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität
München zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.
Vorsitzender: Univ.-Prof. Dr. M. Schuster
Prüfer der Dissertation: 1. Univ.-Prof. Dr. R. Niessner
2. Univ.-Prof. Dr. P. Schieberle
Die Dissertation wurde am 11.04.2011 bei der Technischen Universität München eingereicht
und durch die Fakultät für Chemie am 07.07.2011 angenommen.












Für Lars

This work was made possible by the generous funding of the Federal Ministry of Education
and Research (BMBF, project No. 0315036) and of Eurofins Wej Contaminants GmbH. The
experimental work was conducted under the kind supervision of Prof. Dr. Reinhard Niessner
during the time comprising August 2008 to July 2010.
Part of the research presented in this work has already been published:
J.C. Sauceda-Friebe, Xaver Y.Z. Karsunke, Susanna Vazac, Scarlett Biselli, Reinhard
Niessner, Dietmar Knopp, Regenerable immuno-biochip for screening ochratoxin A in green
coffee extract using an automated microarray chip reader with chemiluminescence detection,
Anal. Chim. Acta 2011, 689(2), 234-242.
During her doctoral work, the author collaborated in the following original scientific papers:
Z. Lin, J.C. Sauceda-Friebe, J. Lin, R. Niessner, D. Knopp, Double-codified nanogold
particles based automated flow-through CLEIA for 2,4-dinitrotoluene Anal. Methods 2010,
2(7), 824-830.
D. Tang, J.C. Sauceda, Z. Lin, S. Ott, E. Basova, I. Goryacheva, S. Biselli, J. Lin, Magnetic
nanogold microspheres-based lateral flow immunodipstick for rapid detection of aflatoxins in
food, Biosens. Bioelectron. 2009, 25(2), 514-518.
M. Rieger, C. Cervino, J.C. Sauceda, R. Niessner, D. Knopp, Efficient hybridoma screening
technique using capture antibody based microarrays, Anal. Chem. 2009, 81(6), 2373-2377.
C. Cervino, J.C. Sauceda, R. Niessner, D. Knopp, Mycotoxin analysis by automated flow-
through immunoassay with chemoluminescence readout, Luminescence 2008, 23(4), 206-207.
I would like to express my heartfelt thanks to my mentor, Prof. Dr. Reinhard Niessner, for the
interesting topic and the uninterrupted support offered during the time of my PhD. I would
also like to thank Prof. Dr. Dietmar Knopp for the fruitful discussions and the generous
guidance in the realization of this project.

My gratitude also goes to Xaver Karsunke, Susanna Vazac, Dr. Philipp Stolper, Dr. Christian
Cervino, and Dr. Sung Zhe, for the memorable times, the invaluable help, and the excellent
group atmosphere that made my stay at the Institute unforgettable.

To Dr. Natalia Ivleva I thank for her help with the microscopy pictures and for much, much
more.

I am thankful to Klaus Wutz for the critical review of my scientific article and for the
excellent discussion opportunities.

To my colleagues and friends from the IWC community I am indebted because they were
always there for me when I most needed them, for a good time, or a good laugh, for insightful
advice or a helping hand.

The students that worked under my supervision earned a permanent place in my heart.
Without them, this work would not have been possible.

I would like to thank Dr. Jürgen Groll for the generous samples of Star-PEG prepolymeres.

Dr. Michael Weller has my gratitude for the helpful and friendly advice that took me a step
further in the experimental research.

I would also like to thank Dr. Scarlett Biselli of Eurofins WEJ Contaminants and her helpful
team for providing the mycotoxin contaminated food samples and the official mycotoxin
norms.

Finally, I am indebted to my family, Rocío, Juan Manuel, Regina, Roland, Patricia and
Daniel, for this wonderful place that I take always with me, which is called Home. Table of Contents

1 Introduction and Problem Stating.................................................................................. 1
2 Theoretical Background .................................................................................................. 3
2.1 Ochratoxin A: Relevance and Analytics ................................................................ 3
2.1.1 Mycotoxins: Definition, Origin and Importance................................................ 3
2.1.2 OTA, Generalities.............................................................................................. 5
2.1.3 OTA, Toxicity Assessment ................................................................................ 6
2.1.4 OTA Contamination in Foods: Impact and Legislation..................................... 7
2.1.5 Mycotoxin Analytics: Challenges and Trends ................................................... 9
Sample and sample preparation........................................................................................ 10
Extraction ......................................................................................................................... 10
Sample Clean-up .............................................................................................................. 11
Detection .......................................................................................................................... 15
2.2 Chemical Immobilization of Receptors on Biosensors........................................ 25
2.2.1 Diversity and Challenges in Sensor Miniaturization 25
2.2.2 Physical Adsorption......................................................................................... 26
2.2.3 Covalent Immobilization.................................................................................. 26
2.2.4 Receptor Immobilization: Affinity Binding..................................................... 31
2.2.5 Receptor Immobilization: Analyte Derivatization ........................................... 32
2.2.6 Arraying Methods: Analyte Deposition Techniques........................................ 34
2.3 Solid Phase Peptide Synthesis (SPPS) .................................................................. 36
2.3.1 Generalities and Principle of SPPS 36
2.3.2 Activating Reagents in Fmoc Chemistry ......................................................... 39
2.3.3 SPPS Solid Supports ........................................................................................ 41
2.3.4 Side Reactions in SPPS.................................................................................... 42
Racemization at the α-Carbon.......................................................................................... 42
i Diketopiperazine Formation............................................................................................. 43
Aspartimide Formation .................................................................................................... 43
3 Results and Discussion................................................................................................... 45
3.1 Customization and Characterization of the MCR 3 ........................................... 45
3.1.1 MCR 3: General Description and Characteristics ............................................ 45
3.1.2 The Flow-Cell Work Surface ........................................................................... 50
3.1.3 Analyte Chemical Modification for Immobilization........................................ 52
3.1.4 Reactive Surface Characterization: Contact Spotting ...................................... 55
3.1.5 Optimization of Surface Regeneration Conditions .......................................... 56
3.1.6 Signal Decrease with Surface Regeneration .................................................... 60
3.1.7 Additional Surface Modifications: Star-PEG................................................... 63
3.1.8 Instrumentation: Flow Cell Characteristics...................................................... 65
3.1.9 Instrumentation: Optimized Assay Conditions ................................................ 68
3.2 MCR 3-Based OTA Determination in Green Coffee .......................................... 71
3.2.1 HPLC Testing of Blank Green Coffee ............................................................. 71
3.2.2 Comparison of Four Available Anti-OTA Antibodies..................................... 75
3.2.3 Optimization of Assay Conditions with Coffee Extract................................... 78
3.2.4 MCR 3 OTA Measurements in Green Coffee Extract: Search for an Adequate
Positive Control................................................................................................................ 80
3.2.5 Dose-Response Curves in Buffer and in Green Coffee Extract....................... 83
3.2.6 Real Sample Measurements ............................................................................. 91
3.2.7 Comparison of the Developed MCR 3 Method with Other Available Methods
for OTA Screening in Coffee ..................................................