Automated System for the Cell-Free Protein Microarray Synthesis and the Label-Free Molecule-Protein Interaction Analysis

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Automated System for the Cell-Free Protein Microarray Synthesis and the Label-Free Molecule-Protein Interaction Analysis Automated system for the cell-free protein microarray synthesis and the label-free molecule-protein interaction analysis Dissertation zur Erlangung des Doktorgrades der Technischen Fakultät der Albert-Ludwigs-Universität Freiburg vorgelegt von Jürgen Burger Freiburg im Breisgau, Juli 2017 Tag der mündlichen Prüfung: 28.11.2017 Dekan Prof. Dr. Oliver Paul (Universität Freiburg) Referenten Prof. Dr. Gerald Urban (Universität Freiburg) Prof. Dr. Günter Gauglitz (Universität Tübingen) Betreuer Dr. Günter Roth (Universität Freiburg) Erklärung Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quelle gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche Hilfe von Vermittlungs- oder Beratungsdiensten (Promotionsberaterinnen oder Promotionsberatern oder anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen. Die Arbeit wurde bisher weder im In- noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt. Ich erkläre hiermit, dass ich mich noch nie an einer in- oder ausländischen wissenschaftlichen Hochschule um die Promotion beworben habe oder gleichzeitig bewerbe. __________________________________________ Datum/Date Unterschrift / Signature iii Abstract Protein microarrays are essential tools for high-throughput proteome interaction and binding kinetics analysis. Recent technical developments have resulted in approaches for the generation of protein arrays by in situ cell-free expression of DNA array templates [1]. We combined and enhanced these ideas by creating innovative microfluidic flow cells in standard microscope slide format and such improved system efficiency, operability, robustness and controllability. Two of these flow cell designs proved to be most suitable, the booklet design and the structured PDMS slide design. Both incorporate the idea of having a highly parallel microfluidic gap of ~ 30 µm between a template DNA microarray and a protein capture surface. The in situ protein expression is initiated by priming the microfluidic gap with ~ 20 μl of cell-free protein expression mix, after an incubation of 30 to 90 minutes at a temperature of ~ 37 °C the protein microarray is created. Despite free diffusion of the proteins they mainly immobilize highly specific by fusion- tag as rather sharp-edged spots opposite of their DNA spot origin, such creating a protein image of the DNA array. Compared to the most advanced cell-free in situ protein microarray generation technique DAPA [2], we obtained protein copies of similar quality however, the system robustness could be significantly enhanced. Furthermore, the microfluidic system decreases the consumption of the rather expensive cell-free system to one-fifth and cut down the copy process time to half. Our new system yields easy and quick operability with slide exchange times of 1-2 minutes versus > 10 minutes for DAPA. After a first proof-of-concept for the in situ expression of protein microarrays in our newly devised microfluidic flow cells, an automated setup suitable for both the in situ expression as well as the interaction analysis of protein microarrays by flow-injection was designed, built and evaluated. The reflecto-interferometric principle patented by Biametrics GmbH, Tübingen, Germany, was selected to enable a label-free imaging detection. Such, major deficiencies of most prominent methods for imaging label-free interaction analyses could be circumvented: sensitivity of SPR against temperature drifts and plasmon crosstalk; low number of parallel measurements (< 384) typical for imaging surface plasmon resonance (SPRi), quartz crystal microbalance (QCM) or bio-layer interference (BLI). Aside label-free detection the automated system provides fluidic and temperature steering elements controlled by a programmable process protocol defining parameters for assays with up to 44 reagents. Matlab based software enables automated spot identifications followed by evaluations of these spots resulting in binding curves and finally the determination of binding constants. The total system is validated physically with a bBSA Streptavidin based assay. System sensitivity with baseline noise of < 3 × 10–9 RSS/min and baseline drift of < 0.003 ‰/min proves to be suitable for various biochemical iv assays. Spotting pattern analyses revealed effects of reagent depletion depending both on concentrations of ligand or reagents as well as spot geometry. Due to available spotting technologies microarrays have been analyzed with a spatial resolution of 2 spots/mm2 (max. 540 spots for applied array of size 18 mm x 15 mm); up to 600 spots/mm2 , in total 162,000 spots at a spatial resolution of ~ 40 µm could be detected. Further biochemical system evaluation by thrombin aptamer assays resulted in a KD of ~ 100 nM, which is in correspondance to literature values. Various antibody antigene assays and a rabbit sera immunization assay were proving this system to be an efficient tool for interaction screenings. Finally both, the protein microarray expression followed by the microarray interaction analysis was performed sequently in the same microfluidic flow cell. Such the created protein spots have never been in contact with air: this is a major advantage, by now only realized in the PING (Protein In situ Network Generator) chip, a large scale microfluidic integration chip, which is significantly more complex to built and less robust to work with. Moreover, with our microfluidic system multiple expressions of a single DNA array with remarkable reproducibilities have been performed in 2017 by Normann Kilb and Tobias Herz, both working in our research group. Next the following topics will be tackled for improving the system additionally: detailed protein expression analyses considering diffusion processes and depletion effects upon various DNA spotting patterns with different concentrations, densities, geometries. improving flow cell morphology for a more homogeneous flow on the microarray. increasing SNR by enhancing the optical system with modified reflective layers to enable the detection and analysis of low molecular binders or very weak interactions. automation of washing procedures for further increasing system robustness and overall process reproducibility. deepening biochemical analysis of protein synthesis, e.g. codon usage, IRES sites, transcription factors, ribosome stalling. expanding applications to the screening of phage / ribosome displays or pre-defined DNA arrays for allergens analysis and the determination of vaccination status or autoimmunities. Considering all aspects being realized or being latently available, this system has a huge potential for both high-density cell-free protein microarray synthesis and versatile high-throughput proteomic analyses. v Zusammenfassung Mikroarrays sind ein Grundpfeiler der Hochdurchsatz-Proteom-Interaktionsanalyse und unerlässlich bei der quantitativen Analyse von Bindungskinetiken. Zu den neuesten technologischen Entwicklungen gehören vor allem die Methoden zur direkten Generierung von Protein-Arrays aus DNA-Arrays mittels zellfreier in situ Expression [1]. Diese diversen Lösungsansätze haben wir nun kombiniert und innovativ erweitert, indem wir mikrofluidische Flusszellen im Standard-Objektträgerformat entwickelten, die ein System mit höherer Effizienz, verbesserter Handhabbarkeit, Robustheit und Regelbarkeit realisierten. Zwei der Designentwürfe haben sich als besonders gelungen erwiesen, das Booklet Design und das Design mit einem strukturierten PDMS Slide. Beide Designentwürfe basieren auf der Idee, einen präzise parallelen, mikrofluidischen Spalt mit ~ 30 µm Höhe zwischen dem DNA-Mikroarray und der Protein- Fängeroberfläche zu schaffen. Die in situ Proteinsynthese beginnt zeitlich exakt definiert mit dem Befüllen des mikrofluidischen Spaltes mit ~ 20 µl des zellfreien Proteinsynthesesystems. Nach einer 30 – 90 minütigen Inkubation bei einer Temperatur von ~ 37 °C ist das Protein Mikroarray hergestellt. Trotz der freien Diffusion der Proteine immobilisieren diese mittels eines Fusions- Tags hochspezifisch als bemerkenswert scharfkantige Spots gegenüber ihrer originären DNA Spots, somit wird ein Proteinabbild des DNA Arrays geschaffen. Im Vergleich zu DAPA [2], der wohl fortschrittlichsten Methode zur Erzeugung von Protein-Mikroarrays aus einem DNA- Mikroarray, konnten wir Proteinkopien von vergleichbarer Qualität erzeugen, die Robustheit des Systems jedoch deutlich verbessern. Darüber hinaus hat das entwickelte mikrofluidische System den Verbrauch teurer Biochemikalien auf ein Fünftel reduziert und die Zeit zur Erzeugung einer Protein-Kopie halbiert. Unser System ist einfach und schnell zu handhaben, einer Objektträger- Wechselzeit von 1-2 Minuten stehen mehr als 10 Minuten bei DAPA gegenüber. Nach einem ersten Machbarkeitsnachweis für die Synthese von Protein-Mikroarrays in einer mikrofluidischen Flusszelle wurde ein komplett automatisiertes Gerät für diese Protein-Synthese und die nachfolgende Interaktionsanalyse mittels Echtzeit-Kinetik-Messungen entworfen, gebaut und getestet. Das reflekto-interferometrische Prinzip (patentiert von Biametrics GmbH, Tübingen) wurde hierbei als label-freies Detektionssystem ausgewählt, da es wesentliche
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