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Process Development for Manufacturing Stochastic Peptide Microarrays Process Development for Manufacturing Stochastic Peptide Microarrays Zur Erlangung des akademischen Grades eines DOKTORS DER INGENIEURSWISSENSCHAFTEN (Dr.-Ing.) von der KIT-Fakultät für Maschinenbau des Karlsruher Instituts für Technologie (KIT) angenommene DISSERTATION von M.Sc. Roman Popov Tag der mündlichen Prüfung: 20. März 2018 Hauptreferent: apl. Prof. Dr. Alexander Nesterov-Müller Korreferenten: Prof. Dr. Jan Korvink Prof. Dr.-Ing. Matthias Franzreb Dieses Werk ist unter einer Creative Commons Namensnennung – Nicht-kommerziell – Weitergabe unter gleichen Bedingungen 4.0 International Lizenz (CC BY-NC-SA 4.0) lizensiert: https://creativecommons.org/licenses/by-nc-sa/4.0/deed.de This document is licensed under a Creative Commons Attribution- NonCommercial-ShareAlike 4.0 International License (CC BY-NC-SA 4.0): https://creativecommons.org/licenses/by-nc-sa/4.0/deed.en Abstract Tackling the challenges of developing therapies for cancer, infections, or immune system disorders requires understanding and manipulation of the metabolic pathways related to a disease state or a pathogen. An essential role in the corresponding biochemical cascades is played by proteins of various types, whose functions are manifested through their interactions with other molecules. In basic and applied research, the functionality and binding properties of proteins are more often studied using peptide microarrays, which are collections of protein fragments displayed on a solid support in a spot array format. Commercially available peptide microarrays are manufactured using various methods, which result in different spot densities and costs per spot. While being relatively simple and straightforward to implement, the wide-spread SPOT- technique provides less than a thousand of peptides on a standard size substrate, which is not sufficient for many biological applications. At the other extreme, the lithographic method enables the synthesis of several million spots per substrate, however, at high manufacturing costs, which makes them unaffordable for many researchers. Thereby, the market need for high-density peptide microarrays at a moderate price remains still unmet. Within the framework of the present work, a new method for manufacturing low- cost high-density peptide microarrays was developed and optimized. Successful project implementation resulted in a novel type of peptide microarray, which is referred in the present dissertation as a stochastic peptide microarray. It contains nearly 3 million synthetic peptides per substrate at material costs of around €250. This was made possible by combining the basic principles and methods of microstructure technology, solid-phase peptide synthesis, and the phenomenon of self-organization of microbeads on a microstructured substrate. i Kurzfassung Um die Herausforderungen der Entwicklung von Therapien gegen Krebs, Infektionen oder Störungen des Immunsystems zu bewältigen, müssen die Stoffwechselwege, die mit einem Krankheitszustand oder einem Krankheitserreger in Zusammenhang stehen, verstanden und manipuliert werden. Eine wesentliche Rolle in den entsprechenden biochemischen Kaskaden spielen Proteine verschiedener Art, deren Funktionen sich in Wechselwirkungen mit anderen Molekülen manifestieren. In der Grundlagen- und angewandten Forschung werden die Funktionalität und die Bindungseigenschaften von Proteinen immer häufiger unter Verwendung von Peptidmikroarrays untersucht, bei denen es sich um Sammlungen von Proteinfragmenten handelt, die auf einem festen Träger in einem Spot-Array-Format immobilisiert werden. Auf dem Markt erhältliche Peptidmikroarrays werden mit verschiedenen Methoden hergestellt, die zu unterschiedlichen Spotdichten und Kosten pro Spot führen. Obwohl sie relativ einfach und unkompliziert zu implementieren ist, liefert die weit verbreitete SPOT-Technik weniger als tausend Peptide auf einem Objektträger, was für viele biologische Anwendungen nicht ausreichend ist. Im anderen Extrem ermöglicht das lithographische Verfahren die Synthese von mehreren Millionen Spots pro Substrat, jedoch bei hohen Herstellungskosten, was sie für viele Forscher unerschwinglich macht. Dadurch bleibt der Marktbedarf nach hochdichten Peptidmikroarrays zu einem moderaten Preis unerfüllt. Im Rahmen der vorliegenden Arbeit wurde ein neues Verfahren zur Herstellung von kostengünstigen hochdichten Peptidmikroarrays entwickelt und optimiert. Die erfolgreiche Projektrealisierung führte zu einem neuartigen Peptidmikroarray, das in der vorliegenden Arbeit als stochastisches Peptidmikroarray bezeichnet wird. Es enthält fast 3 Millionen synthetische Peptide pro Substrat bei Materialkosten von rund 250 €. Möglich wurde dies durch die Kombination der Grundlagen und Methoden der Mikrostrukturtechnik und der Festphasen-Peptidsynthese, sowie auch des Phänomens der Selbstorganisation von Mikropartikeln auf einem mikrostrukturierten Substrat. iii Table of Contents List of Figures ......................................................................................................................... ix List of Tables ....................................................................................................................... xiii List of Abbreviations and Symbols .................................................................................. xv 1 Introduction ..................................................................................................................... 1 1.1 Background and Context ......................................................................................... 1 1.1.1 Proteins and Peptides........................................................................................ 1 1.1.2 Peptide Microarrays .......................................................................................... 4 1.1.3 Solid-Phase Peptide Synthesis ......................................................................... 6 1.1.4 State-of-the-Art Manufacturing Methods .................................................... 10 1.2 Motivation and Problem Formulation ................................................................. 17 1.3 Structure of Dissertation ........................................................................................ 19 2 Stochastic Peptide Microarrays .................................................................................. 21 2.1 Manufacturing Concept ......................................................................................... 22 2.2 Process Steps: Requirements and Implementation ............................................ 26 2.2.1 Substrate Manufacturing ................................................................................ 26 2.2.2 Microbead Manufacturing ............................................................................. 28 2.2.3 Microbead Deposition ..................................................................................... 33 2.2.4 Image Acquisition ............................................................................................ 37 2.2.5 QD Label Decoding ......................................................................................... 40 2.2.6 Amino Acid Extraction and Coupling .......................................................... 41 2.2.7 Microbead Removal ........................................................................................ 44 2.3 Theoretical Foundations ........................................................................................ 46 2.3.1 Adhesion of Microbeads ................................................................................. 46 2.3.2 Excitation and Emission of Quantum Dots.................................................. 48 v Table of Contents 2.3.3 DBSCAN Clustering ....................................................................................... 50 2.3.4 Capillary Condensation ................................................................................. 51 2.3.5 Diffusion of Amino Acid Derivatives .......................................................... 52 3 Materials and Methods ............................................................................................... 55 3.1 Functionalized Substrates ..................................................................................... 55 3.1.1 Microstructured Substrates............................................................................ 55 3.1.2 Flat Substrates .................................................................................................. 59 3.1.3 Quality Control ................................................................................................ 60 3.2 Microbeads .............................................................................................................. 60 3.2.1 Architecture and Composition ...................................................................... 60 3.2.2 Microbead Manufacturing ............................................................................. 61 3.2.3 Optimization and Quality Control ............................................................... 64 3.3 Microparticle Deposition ....................................................................................... 65 3.4 Image Acquisition .................................................................................................. 66 3.5 QD Label Decoding ................................................................................................ 68 3.6 Amino Acid Extraction and Coupling................................................................
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