Conception and Fabrication of Reusable Microfluidic Tools to Study
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NNT : 2017SACLS226 THESE DE DOCTORAT DE L’U NIVERSITE PARIS -SACLAY PREPAREE A L’UNIVERSITE PARIS -SUD ECOLE DOCTORALE N° 575 Electrical, o ptical, bio-physics and engineering Spécialité de doctorat : Electronique et Optoélectronique, Nano- et Microtechnologies Par Xuan ZHAO Conception and fabrication of reusable microfluidic tools to study the dynamics of biological phenomena: application to antibiotic influx/efflux in bacteria and to cell migration during mouse development Thèse présentée et soutenue au C2N Orsay, le 7 Septembre 2017 : Composition du Jury : M. Bruno LE PIOUFLE Professeur ENS Cachan, Laboratoire SATIE Président M. Vincent STUDER DR CNRS , Institut Interdisciplinaire de Neurosciences Rapporteur Mme Marie-Caroline JULLIEN DR CNRS, Laboratoire Gulliver ESPCI Rapporteur M. Pierre-Henri PUECH CR INSERM , Laboratoire Adhésion et Inflammation Examinateur M. Charlie GOSSE CR CNRS , Centre de Nanosciences et de Nanotechnologies Directeur de thèse Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l’Orme aux Merisiers RD 128 / 91190 Saint -Aubin, France Acknowledgements My first thanks will be given to Bruno Le Pioufle, Vincent Studer, Marie-Caroline Jullien, and Pierre-Henri Puech, members of the committee who have accepted to read my PhD manuscript and judge my work. Secondly, I would like to express my sincere gratitude to my thesis advisor, Charlie Gosse (C2N Marcoussis, CNRS), for his kind encouragement, clear guidance, great patience, and continuous support throughout the whole research project. His comprehension and knowledge of microfluidics, microfabrication, and numerical simulations were extremely valuable for improving the quality of my thesis and of the publications that have been written. His insightful comments and suggestions helped me to make progresses in my microfluidic studies, in the redaction of the dissertation as well as in the capture of my future career. I am also extremely grateful to Matthieu Réfrégiers (DISCO beamline, SOLEIL Synchrotron) for his invitation and for the permission to conduct my research on the TELECOM microscope. My integration in the beamline team and the financial support I have benefited for my work were much valuable. I also appreciated all the members of the Biology Laboratory and of the Chemistry Laboratory of the SOLEIL Synchrotron for their help and assistance during my experimentals. I am grateful to my collaborators, Aitana Perea-Gomez, Isabelle Migeotte, Diana Suárez-Boomgaard, Joni Frederick, Julia Vergalli, and Anne-Marie Tran, for their help and for the harmonious cooperation during my thesis period. I want to express my deep appreciation to my friends and colleagues for their support: Elsa Mazari, Anne-Claire Loüer, Hugo Salmon, Antoine Barbot, and Qiongdi Zhang, who were Ph.D students in the same laboratory than me. All the members of the C2N clean room are also deeply acknowledged for their kind guidance on the use of the machines and for the constructive exchanges and discussions we had all these years. Finally, I would like to express my great respect to my family for their warmly encouragement and continuous support. Table of content Part I. A reusable microdevice for dynamic studies of antibiotics uptake in immobilized individual bacterium Chapter I. Various view points on antibiotherapy ___________________________ 11 I. Historical perspective on drug discovery and development _____________________ 11 I.1. Early findings _______________________________________________________________ 11 I.2. The discovery of antagonism between microorganisms _____________________________ 12 I.3. The discovery of penicillin _____________________________________________________ 12 I.4. The golden era ______________________________________________________________ 14 I.4.1. Natural compounds in the 1940s-1950s ______________________________________ 14 I.4.2. Semi-synthetic drugs in the 1960s ___________________________________________ 14 I.4.3. Synthetic quinolones in the 1980s ___________________________________________ 15 I.5. The discovery void ___________________________________________________________ 15 II. Public health issues related to drug resistance ______________________________ 16 II.1. Emergence of the phenomena and anticipated consequences _______________________ 16 II.2. Reported causes ____________________________________________________________ 17 II.2.1. Endopathic factors _______________________________________________________ 17 II.2.2. Exopathic factors ________________________________________________________ 19 II.3. Possible solutions ___________________________________________________________ 20 II.3.1. Rational use of antibiotics by doctors and patients _____________________________ 20 II.3.2. Increase supervision and support by policy makers _____________________________ 20 III. Cellular and molecular aspects __________________________________________ 21 III.1. Classification of bacteria according to their structure ______________________________ 21 III.1.1. Appearance and shape ___________________________________________________ 21 III.1.2. Result of Gram staining __________________________________________________ 22 III.2. Classification of antibiotics according to their target _______________________________ 23 III.2.1. Inhibition of cell wall synthesis ____________________________________________ 24 III.2.2. Disruption of the cytoplasmic membrane ____________________________________ 25 1 / 192 III.2.3. Inhibition of folate synthesis ______________________________________________ 25 III.2.4. Inhibition of protein synthesis _____________________________________________ 26 III.2.5. Inhibition of nucleic acids synthesis _________________________________________ 27 III.3. Classification of resistances according to their mechanism __________________________ 28 III.3.1. Drug inactivation by an enzyme ____________________________________________ 29 III.3.2. Alteration of the target site _______________________________________________ 29 III.3.3. Changes of membrane permeability ________________________________________ 30 III.3.4. Active expulsion of the drug from the bacteria ________________________________ 30 III.4. Description of the biomolecules involved in drug influx/efflux _______________________ 30 III.4.1. The cell wall of the Gram-negative bacteria __________________________________ 30 III.4.2. The OmpC and OmpF porins ______________________________________________ 31 III.4.3. The multi drug efflux pumps ______________________________________________ 32 Chapter II Strategies used for influx/efflux measurements ___________________ 35 I. A review of the literature ________________________________________________ 35 I.1. Bulk studies_________________________________________________________________ 36 I.1.1 Natural systems __________________________________________________________ 36 I.1.2 Artificial systems _________________________________________________________ 37 I.2. Single cell studies ____________________________________________________________ 38 I.2.1. Natural systems _________________________________________________________ 38 I.2.2 Artificial systems _________________________________________________________ 39 I.3 Single protein studies _________________________________________________________ 40 II. Our biological system __________________________________________________ 41 II.1. Escherichia coli , a typical Gram-negative bacteria __________________________________ 42 II.1.1. Main characteristics______________________________________________________ 42 II.1.2. Relevance for public health ________________________________________________ 43 II.1.3. Use as a model organism __________________________________________________ 43 II.2. Quinolones, a family of fluorescent antibiotics ____________________________________ 44 II.2.1. Structural properties _____________________________________________________ 44 II.2.2. Mechanism of action _____________________________________________________ 45 II.2.3. Relevance for public health ________________________________________________ 46 II.2.4. Spectroscopic properties __________________________________________________ 47 III. The experiments performed at SOLEIL ____________________________________ 47 III.1. Intrinsic fluorescence imaging of antibiotic concentration __________________________ 48 III.1.1. Necessity of a real-time label-free technique _________________________________ 48 III.1.2. Description of the Telemos epifluorescence microscope ________________________ 49 III.1.3. Specific issues related to UV fluorescence observation _________________________ 51 2 / 192 III.2. Previous protocol ___________________________________________________________ 52 III.2.1. Description ____________________________________________________________ 52 III.2.2. Limitations ____________________________________________________________ 54 III.3. Proposed protocol __________________________________________________________ 54 Chapter III. Design and fabrication of the microdevice _______________________ 55 I. Criteria to be fulfilled ___________________________________________________ 55 I.1. Complete cell immobilization __________________________________________________ 55 I.2. Parallel monitoring of individual bacteria _________________________________________ 56 I.3. Quick change of the chemical environment _______________________________________ 56 I.4. Practicity ___________________________________________________________________ 57 II. Existing technologies ___________________________________________________