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Terbium Nanoparticle Biofunctionalization for Extracellular Biosensing Vjona Çifliku

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Terbium Nanoparticle Biofunctionalization for Extracellular Biosensing Vjona Çifliku Terbium nanoparticle biofunctionalization for extracellular biosensing Vjona Çifliku To cite this version: Vjona Çifliku. Terbium nanoparticle biofunctionalization for extracellular biosensing. Inorganic chem- istry. Université Paris-Saclay, 2020. English. NNT : 2020UPASS075. tel-02938737 HAL Id: tel-02938737 https://tel.archives-ouvertes.fr/tel-02938737 Submitted on 15 Sep 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Terbium nanoparticle biofunctionalization for extracellular biosensing Thèse de doctorat de l'université Paris-Saclay École doctorale n°575: Electrical, optical, bio: physics and engineering (EOBE) Spécialité de doctorat: Physique (Electronique et Optoélectronique, Nano et Microtechnologies) Unité de recherche : Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France Référent : Faculté des sciences Thèse présentée et soutenue à Orsay, le 9 Juin 2020, par Vjona Çifliku Composition du Jury Sandrine LACOMBE Présidente Professeure, Université Paris Saclay Alexandra FRAGOLA Rapporteur & Examinatrice Maîtresse de Conférence, Sorbonne Université SS075 Peter REISS Rapporteur & Examinateur Chercheur, CEA Grenoble Loïc CHARBONNIÈRE Examinateur Chercheur, CNRS Strasbourg : 2020UPA NNT Niko HILDEBRANDT Directeur de thèse Thèse de doctorat de Thèse Professeur, Université Paris Saclay ECOLE DOCTORALE N°575 Electrical, optical, bio: physics and engineering (EOBE) Titre : Bio fonctionnalisation des nanoparticules de terbium pour la bio détection extracellulaire Mots clés : Nanoparticules de lanthanides, détection à résolution temporelle, microscopie à luminescence, EGFR, bioconjugaison Résumé : Les nanoparticules dopées au lanthanide des lignées cellulaires exprimant fortement (A431) (LnNPs) sont devenues une classe importante de et faiblement (HeLa et Cos7) l'EGFR, alors que les fluorochromes pour la biodétection et la bioimagerie lignées cellulaires non exprimantes (NIH3T3) et les avancées. Cependant, le développement de LnNP cellules A431 passives de l'EGFR n'ont montré brillants, stables et bioconjugués reste un défi aucun signal. Malgré la taille relativement important pour leur traduction en applications importante des conjugués TbNP-anticorps, ils ont bioanalytiques fonctionnelles. Ici, nous présentons la pu être internalisés par les cellules A431 lors de leur détection in situ de la liaison ligand-récepteur sur les liaison à l'EGFR extracellulaire, qui a montré leur cellules a été accompli avec des conjugués TbNP- potentiel en tant que marqueurs de luminescence anticorps (Matuzumab) qui pourraient se lier brillants et stables pour la signalisation Des tests de spécifiquement aux récepteurs transmembranaires stabilité des TbNP sur une gamme de valeurs de du facteur de croissance épidermique (EGFR). Une pH, de tampons différents, dans le temps, et d'effet spécificité et une sensibilité élevées ont été de sonication sur ces NPs ont été effectués en démontrées par l'imagerie temporelle de l'EGFR sur utilisant la DLS et la ZP. Title : Terbium nanoparticle biofunctionalization for extracellular biosensing Keywords : Lanthanide nanoparticles, time-resolved detection, luminescence microscopy, EGFR, bioconjugation Abstract: Lanthanide-doped nanoparticles (LnNPs) (HeLa and Cos7) EGFR-expressing cell lines, have become an important class of fluorophores for whereas non-expressing cells did not show any advanced biosensing and bioimaging. However, signals. Despite the relatively large size of TbNP- developing bright, stable, and bioconjugated LnNPs antibody conjugates, they could be internalized by remains an important challenge for their translation A431 cells upon binding to extracellular EGFR, into functional bioanalytical applications. Here, we which showed their potential as bright and stable present in-situ detection of ligand-receptor binding luminescence markers for intracellular signaling. on cells, which was accomplished with TbNP- Stability tests of TbNPs over a range of pH values, antibody (Matuzumab) conjugates that could different buffers, over time, and sonication effect specifically bind to transmembrane epidermal on these NPs were performed by using dynamic growth factor receptors (EGFR). High specificity and light scattering (DLS) and zeta potential (ZP) sensitivity were shown by time-gated imaging of measurements. EGFR on both strongly (A431) and weakly Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l’Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Acknowledgment In the first place, I wish to thank a lot from the bottom of heart my supervisor Prof. Dr. Niko Hildebrandt for his fundamental role and support in my doctoral work. I am very grateful for his trust in my abilities to carry on with the project with total freedom! He gave me the opportunity to follow my professional dreams and supported me in every step. I also would like to thank him for the great German beer and Italian coffee. I thank a lot MCF Alexandra Fragola and Dr. Peter Reiss for being the reviewers of my Ph.D. thesis. I appreciate a lot their time spent reading my thesis. I also wish to thank our collaborators Loïc Charbonniere, Cyrille Charpentier, Peter Reiss, and Davina Moodelly for their kindness, availability and scientific discussions we had during NEUTRINO’s meetings. I wish also to thank the group of Philippe Minard (Modélisation et Ingénieur des proteins) for their support and hospitality in the last couple of months of my thesis. Many thanks to all former members who I encountered and current members of the NanoBioPhotonics group for their encouragement, moral and professional support. My special thanks to Dr. Thomas Plénat and Dr. Marcelina Cardoso Dos Santos, for their continuous guidance, patience and valuable time devoted to sharing their technical and scientific knowledge with me. I look up to them as I admire them as great people and great scientists. I would like to thank Dr. Shashi Bhuckory, Dr. Xue Qiu and Dr. Akram Yahia-Ammar for taking care of me when I first arrived at the lab. Special thanks to Mme. Meryem Sennad for her positive attitude towards life, and her friendship! I want to express my gratitude to Misses Jingyue Xu, Mimoza Reçi, and Kleni Mulliri for their unconditional friendships. I gratefully acknowledge the funding sources of my Ph.D. study, National Research Agency (ANR). Finally yet importantly, I am very much indebted to my family and my partner in crime Thomas Plénat, who supported me in every possible way to see the completion of this work. They are my rock! Publications and Communications Publications 1. Ultrabright terbium nanoparticles for FRET biosensing and in-situ imaging of epidermal growth factor receptors. Vjona Cifliku,b‡ Cyrille Charpentier,a‡ Joan Goetz,a,c Aline Nonat,a Marcelina Cardoso Dos Santos,b Laura Francès Soriano,b Ka-Leung Wong,c Niko Hildebrandt,b,* and Loïc J. Charbonnière,a* (Chemistry 2020 Jun 5) 2. Single-Nanoparticle cell Barcoding by Tunable FRET from Lanthanides to Quantum Dots. C. Chen, L. Ao, Y.-T. Wu, V. Cifliku, et al., Angew. Chem. Int. Ed. 2018, 57, 13686. Patent N. Hildebrandt, L.J Charbonnière, V. Cifliku, M. Cardoso Dos Santos, C. Charpentier, J. Goetz, A. Nonat, K.-L. Wong. Ultrabright luminescent lanthanide nanoparticles comprising terbium, with longer excited states. EP 18305890. 07/2018. Oral communications 1. Advanced Courses on Functional Imaging of Nuclear Organization & Signalling (FEBS) at Van Leeuwenhoek Centre for Advanced Microscopy (LCAM) at Amsterdam/Netherlands, Terbium nanoparticle biofunctionalization and characterization for extracellular biosensing [9-15 June 2019] Poster communications 1. 9th International Conference on Nanoscience with Nanocrystals (NaNaX 9) at University of Hamburg/Germany, Terbium nanoparticle biofunctionalization and characterization for extracellular biosensing [16-20 September 2019] 2. Centre National de le Recherche School on Functional Microscopy in Biology (MiFoBio) Seignosse/France, Ultrabright Terbium Nanoparticles for Biosensing [5-12 October 2018] 3. Journée scientifique du département B3S de l’I2BC at auditorium IMAGIF Campus of Centre National de le Recherche at Gif-sur-Yvette/France Ultrabright Terbium Nanoparticles for Biosensing [18 December 2017] Contents 1. Introduction ................................................................ 1 2. Background ................................................................. 6 2.1 Biomarkers ................................................................................................................ 6 2.2 Lanthanides ............................................................................................................ 12 2.2.1 Introduction ...................................................................................................... 12 2.2.2 Luminescence of lanthanide ions .................................................................... 14 2.2.3 Applications of lanthanides ............................................................................
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