Structured antibody surfaces for bio-recognition and a label-free detection of bacteria [Elektronische Ressource] / Cornel Wolf

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2010
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

“Structured Antibody Surfaces for Bio-Recognition and a
Label-free Detection of Bacteria”
Dissertation
zur Erlangung des Grades
„Doktor
der Naturwissenschaften“
am Fachbereich Biologie
der Johannes Gutenberg-Universität
in Mainz
Cornel Wolf
geb. in Erfurt
Mainz, 2010Die vorliegende Arbeit wurde in der Zeit von Januar 2008 bis November 2010 im
Arbeitskreis Materialforschung des Max-Planck-Instituts für Polymerforschung in
Mainz angefertigt.
Dekan:
1. Berichterstatter:
2. Berichterstatter:
Tag der mündlichen Prüfung: 20. Dezember 2010Inmitten der Schwierigkeiten liegt die Möglichkeit.
Albert EinsteinTable of Contents
Summary..................................................................................................................9
Abbreviations.........................................................................................................11
1 General Introduction and Motivation.................................................................13
1.1 Bacteria and Microbial Diseases............................................................................................13
1.2 Established Methods for Pathogen Detection........................................................................15
1.3 Biosensors in Pathogen Detection..........................................................................................18
1.4 Applications of Biosensors.....................................................................................................21
1.5 Desired Properties of Bacterial Biosensors............................................................................21
1.6 Motivation and Aims..............................................................................................................23
2 Materials.................................................................................................................31
2.1 Chemicals and Compounds....................................................................................................31
2.2 Colloidal Crystals...................................................................................................................33
2.3 Inverse Opals (iopals).............................................................................................................43
2.4 Bacteria..................................................................................................................................51
2.5 Bacteria Cultivation................................................................................................................55
2.6 Antibodies..............................................................................................................................56
2.7 Antibody Structures................................................................................................................62
2.8 Integrin Vesicle Formation.....................................................................................................62
3 Methods..................................................................................................................65
3.1 Phase Contrast Microscopy (PCM)........................................................................................65
3.2 Fluorescence Microscopy.......................................................................................................66
3.3 Confocal Laser Scanning Microscopy (CLSM).....................................................................68
3.4 Atomic Force Microscopy (AFM).........................................................................................69
3.5 Scanning Electron Microscopy (SEM)..................................................................................72
3.6 Contact Angle Measurements................................................................................................74
3.7 Wester Blot.............................................................................................................................75
4 Label-free Detection of Microorganisms in an Integrated Biosensor Array...77
4.1 Antibody Specificity against E. coli.......................................................................................77
4.2 Antibody Binding by 3-Aminopropyltriethoxysilane (APTES)............................................79
4.3 Bacteria Binding by Biotin, Streptavidin and NeutrAvidin...................................................87
4.4 Bacteria Binding by Active Active-Ester-Silanes AE-S........................................................91
4.5 Inverse Opals Dimensioning..................................................................................................964.6 Inverse Opals Functionalization...........................................................................................100
4.7 Conclusion and Outlook.......................................................................................................102
5 2D Array-Patterning of Antibody Annuli.........................................................105
5.1 Template Formation.............................................................................................................105
5.2 Protein Patterning.................................................................................................................106
5.3 Antibody Annuli Structures..................................................................................................110
5.4 Proteins Bound to Annuli Structures....................................................................................112
5.5 Negative Control for Specific Binding.................................................................................113
6 Structured Antibody Surfaces for Bio-recognition...........................................115
6.1 Bacteria Whole-Cell Sensing...............................................................................................115
6.2 Cellular Mimics by Integrin Functionalized Lipid Vesicles.................................................119
6.3 P19 Cells Bound to Annuli Structures.................................................................................123
6.4 Circular Antibody Structures...............................................................................................124
6.5 Conclusion and Outlook.......................................................................................................126
7 Size Exclusion Filters..........................................................................................129
7.1 Fabrication Process..............................................................................................................129
7.2 Anti-Crack Approaches........................................................................................................132
7.3 Recrystallized Silicon Carbide Substrates............................................................................134
7.4 200 nm Bacteria-Filters........................................................................................................136
7.5 Conclusion and Outlook.......................................................................................................136
8 General Conclusion and Outlook.......................................................................139
9 Bibliography.........................................................................................................141
10Publications..........................................................................................................161
10.1 Published Manuscripts.........................................................................................................161
10.2 Submitted Manuscripts.........................................................................................................161
11Acknowledgements..............................................................................................163
12Curriculum Vitae.................................................................................................165 Summary
Summary
Antibody microarrays are of great research interest because of their potential application as
biosensors for high-throughput protein and pathogen screening technologies. In this active area,
there is still a need for novel structures and assemblies providing insight in binding interactions
such as spherical and annulus-shaped protein structures, e.g. for the utilization of curved surfaces
for the enhanced protein-protein interactions and detection of antigens. Therefore, the goal of the
presented work was to establish a new technique for the label-free detection of bio-molecules and
bacteria on topographically structured surfaces, suitable for antibody binding.
In the first part of the presented thesis, the fabrication of monolayers of inverse opals with
10 µm diameter and the immobilization of antibodies on their interior surface is described. For this
purpose, several established methods for the linking of antibodies to glass, including Schiff bases,
EDC/S-NHS chemistry and the biotin-streptavidin affinity system, were tested. The employed
methods included immunofluorescence and image analysis by phase contrast microscopy. It could
be shown that these methods were not successful in terms of antibody immobilization and adjacent
bacteria binding. Hence, a method based on the application of an active-ester-silane was introduced.
It showed promising results but also the need for fur