Table of Contents
Chapter 1. Introduction………...………1
1.1 Scope …..…...……….………...1
1.2 Aim of the work……….……….………...…3
1.3 Immobilisation of the probe on the transducer surface ………...…..4
1.3.1 Covalent coupling ... 6
1.3.2 Direct immobilisation of the thiolated probe ... 9
1.4 Electrochemical detection of the recognition event ………11
1.4.1 Label-based electrochemical detection ... 12
1.4.2 Label-free electrochemical detection ... 16
1.5 Organisation of the biomolecules monolayers ……...………21
1.6 Strategy……….……….…..…25
1.6.1 Presentation of the considered system ... 25
1.6.1.1 The p53 tumour protein and the Murine Double Minute 2 oncoprotein ... 25
1.6.1.2 Formation of the MDM2-p53 complex ... 27
1.6.1.3 Electrochemical sensing of MDM2 ... 29
1.6.2 Choice of the electrode material ... 32
1.6.3 Selection of the sequence ... 33
1.6.4 Working plan ... 34
Chapter 2. Experimental ... 35
2.1 Electrochemical techniques……….……...35
2.1.1 The electrochemical cell ... 35
2.1.2 Cyclic voltammetry ... 38
2.1.3 Chronoamperometry and chronocoulometry ... 42
2.1.4 Electrochemical Impedance Spectroscopy ... 43
2.2 Quartz Crystal Microbalance ... 46
2.3 Atomic Force Microscopy ... 48
2.4 In situ fluorescence microscopy ... 49
2.4.1 Experimental setup ... 49
2.5 Chemicals ... 54
Chapter 3. Electrochemical characterisation of the immobilisation of the peptide aptamer probe on gold………... 56
3.1 Formation of the self-assembled monolayers ………...56
3.2 Evidence of the immobilisation of the thiolated molecules………..58
3.3 Interaction between the [Ru(NH3)6]3+ complex and the peptide probe………61
3.3.1 Evidence of the interaction between the complex and the peptide probe ... 61
3.3.2 Influence of the immobilisation procedure on the [Ru(NH3)6]3+ concentration adsorbed at the electrochemical interface………..……….68
3.4 Electrochemical behaviour of the redox couple [Fe(CN)6]3-/4- in presence of the monolayers ... 71
Chapter 4. Elaboration of a p53 peptide-based transducer for the detection of the protein MDM2…...…...….………..72
4.1 Principle of the detection method ………..………...72
4.2 Influence of the contact of the protein MDM2 with the peptide layer on the charge transfer resistance..……….………..………76
4.3 Behaviour of the MDM2 protein on gold and at p53-modified electrodes…………...80
4.4 Quartz crystal microbalance measurements ………..……….84
4.5 Analytical performance ………...………..………...88
4.5.1 Impact of the concentration of MDM2 on the signal………...….88
4.5.2 Negative controls………..………..……….………...90
Chapter 5. Fluorescence microscopy study of self-assembled monolayers of the peptide aptamer probe on gold………..………...94
5.1 Fluorescence spectroscopy ………..…………..………...94
5.1.1 General principles………...……….94
5.1.2 Fluorescence quenching………..………….…………...96
5.1.3 Förster Resonance Energy Transfer……….………..…..………...98
5.1.4 Fluorescence near metal surface…….….………...……….………99
5.1.5 Fluorescence microscopy………….………..……….……..104
5.2 Modification of the peptide probe for fluorescence purposes ………...108
5.3 Interfacial behaviour of the peptide monolayers under polarisation…………...109
5.4 Definition of the regions of interest ………..………....…115
5.5.1 Case of an electrode modified by a two-step adsorption procedure……..……....…118
5.5.2 Case of an electrode modified by a one-step coadsorption procedure………..122
5.6 Interfacial behaviour of a single component layer composed of peptide ……….125
5.7 Study of the heterogeneities of the SAMs ………..………...130
5.8 AFM characterisation of the peptide SAMs ………...134
Chapter 6. Conclusions………..………...139
6.1 General conclusions………. ……….139
