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The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en – Tesi doctoral – INTERDIGITATED ELECTRODE ARRAYS (IDEA) IMPEDIMETRIC TRANSDUCERS FOR BACTERIAL BIOSENSING APPLICATIONS Departament de Genètica i Microbiologia Universitat Autònoma de Barcelona – Doctorat en Biotecnologia – Sergi Brosel Oliu Memòria presentada per Sergi Brosel Oliu per optar al grau de doctor en Biotecnologia per la Universitat Autònoma de Barcelona Vistiplau dels directors: Dr. Andrei Bratov Nikiforov Dra. Natalia Abramova Pavlova Dra. Naroa Uria Moltó Tutor: Dr. Jordi Mas Gordi Institut de Microelectrònica de Barcelona (IMB-CNM, CSIC) Bellaterra, Octubre de 2018 The thesis submitted by Sergi Brosel Oliu, graduate student in Biology and Master degree in Applied Microbiology, entitled Interdigitated electrode arrays (IDEA) impedimetric transducers for bacterial biosensing applications was carried out at Insititut de Microelectrònica de Barcelona (IMB-CNM, CSIC) under the supervision of Dr. Andrei Bratov Nikiforov, Senior Researcher, Dr. Natalia Abramova Pavlova, Researcher, and Dr. Naroa Uria Moltó, Post-Doc Researcher of IMB-CNM. The thesis satisfies the requirements for obtaining the title of PhD in Biotechnology. Part of this work has been performed in the Department of Biomedical Engineering, University Medical Center of Groningen, under the supervision of Dr. Patrick van Rijn, Associate Professor, and Olga Mergel, Post-Doc Researcher. With the approval of: Dr. Andrei Bratov Nikiforov Dr. Natalia Abramova Pavlova Dr. Naroa Uria Moltó Co-directors Dr. Jordi Mas Gordi Sergi Brosel Oliu Tutor Author CONTENTS ABBREVIATIONS, SYMOBOLS AND UNITS __________________________________________________ i SUMMARY __________________________________________________________________________ vii RESUM _____________________________________________________________________________ ix 1. INTRODUCTION _____________________________________________________________________ 1 1.1. Biosensors in the field of microbiology _______________________________________________ 1 1.2. Definition and origins of biosensor technology ________________________________________ 1 1.3. Classification of biosensors ________________________________________________________ 2 1.3.1. Transducer types _____________________________________________________________ 3 1.3.2. Biorecognition elements _______________________________________________________ 3 1.4. Electrochemical biosensors _______________________________________________________ 10 1.5. Electrochemical Impedance Spectroscopy (EIS) fundamentals ___________________________ 11 1.5.1. Interpretation of data and equivalent circuits______________________________________ 13 1.5.2. Faradaic and non-faradaic impedance ___________________________________________ 16 1.6. Interdigitated electrode arrays (IDEA) for impedance measurements: a tool for biosensing ___ 18 1.6.1. Equivalent circuit of IDEAs _____________________________________________________ 19 1.6.2. Three dimensional interdigitated electrode arrays (3D-IDEA) _________________________ 22 1.6.3. Fabrication and characterization of planar IDEA and 3D-IDEA sensors ___________________ 25 1.7. Impedimetric properties of bacterial cells ___________________________________________ 27 1.8. Impedimetric biosensing of bacteria: from impedance microbiology to impedimetric biosensors _________________________________________________________________________________ 29 1.8.1. Impedance biosensors for detection of bacteria ____________________________________ 30 1.8.2. Impedimetric microbial biosensors ______________________________________________ 35 References ________________________________________________________________________ 37 2. OBJECTIVES _______________________________________________________________________ 47 3. METHODS AND RESULTS _____________________________________________________________ 51 3.1. Evaluation of sensitivity and response time of impedimetric transducers modified with polyethyleneimine for bacteria detection _______________________________________________ 51 Abstract ________________________________________________________________________ 51 1. Introduction ___________________________________________________________________ 52 2. Materials and methods __________________________________________________________ 53 3. Results and discussion ___________________________________________________________ 55 4. Conclusions ___________________________________________________________________ 61 References ______________________________________________________________________ 62 3.2. Impedimetric sensor based on 3D interdigitated electrodes for label-free detection of bacterial endotoxins ________________________________________________________________________ 65 Abstract ________________________________________________________________________ 65 1. Introduction ___________________________________________________________________ 66 2. Materials and methods __________________________________________________________ 69 3. Results and discussion ___________________________________________________________ 71 4. Conclusions ___________________________________________________________________ 81 References ______________________________________________________________________ 83 3.3. Performance of a novel and reusable aptasensor for detection of Escherichia coli O157:H7 ____ 87 Abstract ________________________________________________________________________ 87 1. Introduction ___________________________________________________________________ 88 2. Materials and methods __________________________________________________________ 90 3. Results and discussion ___________________________________________________________ 93 4. Conclusions __________________________________________________________________ 100 Supplementary information ________________________________________________________ 100 References _____________________________________________________________________ 105 3.4. 3D impedimetric sensors as a tool for monitoring bacterial response to antibiotics _________ 109 Abstract _______________________________________________________________________ 109 1. Introduction __________________________________________________________________ 110 2. Materials and methods _________________________________________________________ 113 3. Results and discussion __________________________________________________________ 117 4. Conclusions __________________________________________________________________ 126 Supplementary information ________________________________________________________ 127 References _____________________________________________________________________ 129 4. GENERAL DISCUSSION ______________________________________________________________ 135 References _____________________________________________________________________ 140 5. CONCLUSIONS ____________________________________________________________________ 145 General conclusions: _______________________________________________________________ 145 Specific conclusions: _______________________________________________________________ 145 ANNEXES __________________________________________________________________________ 151 Published papers included in this thesis ________________________________________________ 151 Other publications related with this thesis _____________________________________________ 152 ABBREVIATIONS, SYMOBOLS AND UNITS ABBREVIATIONS, SYMBOLS AND UNITS ABBREVIATIONS, SYMOBOLS AND UNITS List of abbreviations 3D-IDEA Three-dimensional Interdigitated electrode arrays AC Alternating current AMP Antimicrobial peptide Amp Ampicillin AST Antimicrobial susceptibility tests ATCC American Type Culture Collection AuNPs Gold nanoparticles BioMEMS Biological microelectromechanical systems BIS N,N´-methylenebis(acrylamide) cross-linker BOD Biological oxygen demand BSA Bovine serum albumin CBPs Carbohydrate binding proteins CD Circular dichroism CLSM Confocal laser scanning microscopy CMOS Complementary metal-oxide-semiconductor CNM Centre Nacional de Microelectrònica Con A Concanavalin A CPE Constant phase element CSIC Consell Superior d’Investigacions Científiques DC Direct Current DEP Dielectrophoresis DEPIM Dielectrophoretic impedance measurements DNA Deoxyribonucleic acid DRIE Deep reactive ion etching E. coli Escherichia coli EC Equivalent circuit EIS Electrochemical Impedance Spectroscopy Fig. Figure GFP Green fluorescent protein Gly Glycogen HMDS Hexamethyldisilazane HRP Horseradish peroxidase HUS Hemolytic uremic syndrome IDEA Interdigitated electrode arrays IMB Institut de Microelectònica de Barcelona ISE Ion selective electrode IUPAC International Union of Pure and Applied Chemistry LAL Limulus amebocyte lysate method LB Luria-Bertani medium LOD Limit of detection LPCVD Low pressure chemical vapor deposition LPS Lipopolysaccharides i IDEA Impedimetric Transducers for Bacterial Biosensing Applications LTA Lipoteichoic acid MBC Minimum bactericidal