Faculté des Sciences
Service de Chimie Analytique et Chimie des Interfaces
Interfacial study of a sensing platform for MDM2,
based on the self-assembly of a p53 peptide on a
gold electrode
Triffaux Eléonore
THESIS SUBMITTED FOR THE DEGREE OF DOCTOR IN SCIENCES
Remerciements
Je tiens à exprimer ma profonde reconnaissance à Madame le Professeur Claudine Buess-Herman pour m’avoir accueillie au sein du Service de Chimie Analytique et Chimie des Interfaces et pour l’intérêt qu’elle a porté à mon travail de rechercher au cours de ces années de thèse. La confiance que vous m’avez témoigné tant sur mon travail de recherche que pour l’enseignement des travaux pratiques m’ont permis de sortir de cette thèse grandie.
Je tiens également à remercier chaleureusement Thomas pour son aide inestimable. Tes connaissances scientifiques, ta disponibilité, ta pédagogie, ta patience et les idées jaillissant sans cesse de ton cerveau ont largement contribué à l’achèvement de ce travail. Ton humour parfois très personnel et le cliquetis de la cuillère dans la tasse carrée auront rythmé cette thèse. Un immense merci!
I also would like to thank Professor Dan Bizzotto from the University of British Columbia for the three months I had the privilege to spend in his lab. I really enjoyed this stay both in a scientific and personal perspective. I am sure that the maple leaves cookies helped me through this work.
Je tiens également à remercier les personnes qui m’ont accompagnée au cours de ma thèse, le Professeur François Reniers, le Professeur Jean-Michel Kauffmann et le Professeur Michele Sferrazza. Leurs remarques judicieuses ont fait avancer ce travail.
J’exprime ma profonde gratitude à François Reniers pour les diverses opportunités qu’il m’a offertes. Ma rencontre avec George Whitesides reste l’un des plus beaux souvenir de ma thèse. Merci!
Un merci tout particulier à Philippe Leclère de l’Université de Mons pour toute l’aide apportée lors des mesures AFM. Ta gentillesse et ta bonne humeur ont fait de ces journées des moments très agréables!
Je me dois bien évidemment de remercier l’ensemble de mes collègues du laboratoire CHANI qui ont rendu ces journées de labo mémorables. Votre bonne humeur et votre soutien pendant la rédaction m’ont beaucoup aidée: Nico, Karim, Greg, Steph V., Quentin, Alp, Perrine, Jonathan, Sami, Jennifer, Roman, Jérika, Francis, Phuong, Anne, Emile, Bernard, Jérémy, Joffrey, Aurore, Titi, Dédé, Julie, Denis, François D., Steph C., Caro, Thomas B., Qiang et Qirong
I also want to thank my colleagues from UBC: Amanda, Jannu and Landis. Thanks a lot for your help!
Merci à Philippe De Keyser et à MacAlbert pour toute l’aide technique fournie et à Sandhya Labouverie pour le support administratif.
ce n’est que votre présence a également illuminé les journées de labo et autres.
Et les vieux de la vieille alors ? Les chimistes des premières heures… Maxence et ton humour douteux, Lionel soulignant ce même humour douteux, Yannick et Steven et leur bienveillance légendaire. La chimie nous a réunis, l’amitié se charge de préserver cette union.
Léo, j’ai toujours pu compter sur toi au long de ces années. Je ne pourrai jamais assez te remercier pour tout ce que tu m’as apporté.
Andrea et Morgane, vous êtes mes valeurs refuges, ma base. A chaque étape, vous êtes présents, jamais vous n’avez failli. Des amis comme vous, il y en a peu et je mesure la chance de vous avoir et le bonheur de pouvoir encore partager tant de moments précieux à vos côtés.
The « best of MCC », qui aurait cru que la valeur ajoutée d’un master complémentaire en gestion en horaire décalé aurait été aussi élevée sur le plan amical. Steph M., Marine, Emilie, Astrid, Elodie, Bryan, Nico et Daniel, grâce à vous le poste immobilisations incorporelles de mon compte de résultats a explosé !
Maman, Papa, Emily, Matthieu, Pèpère et Mèmère aucun mot ne sera suffisant pour vous exprimer ma gratitude. Maman, merci pour ton écoute et tout le réconfort que tu m’as apporté. Mimi et Matt, vous êtes toujours là pour relativiser et me faire rire. Papa, les livres d’or que tu nous lisais m’ont peut-être inconsciemment menée vers cette thèse. Pèpère, tu es parti trop tôt, mais je sais que là où tu es, tu dois être fier. Je vous aime. Maarten, tu es officiellement entré il y a peu dans le clan mais tu t’y es vite intégré. Merci pour ton aide avec les figures. Loulou, ta venue au monde, le jour de mon dépôt de thèse a rendu cette journée inoubliable.
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
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
Abstract
This work focuses on the electrochemical and in situ fluorescence microscopy study of the self-assembly, on gold electrodes, of monolayers of peptide aptamers of the p53 protein for the detection of the protein MDM2. The use of new recognition probes for the molecular recognition such as peptide aptamers has been considered as an alternative to the use of antibodies. Peptide aptamers are synthetic peptide sequences binding the target protein with high affinity and specificity.
The first part of this work consisted in the electrochemical study of the modified interface resulting from different immobilisation procedures. Measurements in the presence of the [Ru(NH3)6]3+ redox marker have evidenced the immobilisation of the peptide probe at the gold
electrode and allowed the relative quantification of the density of probe adsorbed at the electrode with respect to the considered immobilisation procedure. Besides, measurements in the presence of the redox couple [Fe(CN)6]3-/4- showed a dramatic inhibition of the electron
transfer in the case of monolayers exclusively composed of the peptide.
In a second time, we focused on the detection of the protein MDM2. Three modified interfaces were considered, namely two mixed layers of peptide and 4-mercaptobutan-1-ol, the latter playing the role of diluent, adsorbed in one or two step(s), and a monolayer exclusively composed of the peptide. The use of electrochemical impedance spectroscopy in the presence of the redox couple [Fe(CN6)]3-/4- as detection method evidenced the relevance of this latter
interface for the detection. Indeed, the inhibition of the electron transfer previously identified is highly lowered via the interaction with the target protein. A detection range extending from ~1 to 60 ng mL-1 and a limit of detection of 0.69 ng mL-1 have been obtained. This performance can be compared to the commercially available ELISA kits. The reliability and specificity of the response have been tested via negative controls performed on three proteins, namely the fibrinogen, the cytochrome c and the bovine serum albumin and validated through complementary quartz crystal microbalance measurements.
Résumé
Ce travail porte sur l’étude électrochimique et par microscopie de fluorescence in situ de l’auto-assemblage, sur électrode d’or, de monocouches d’aptamères peptidiques de la protéine p53 en vue de la détection de la protéine MDM2. L’utilisation de nouvelles sondes de reconnaissance moléculaire telles que les aptamères peptidiques a été considérée en tant qu’alternative à l’utilisation d’anticorps. Les aptamères peptidiques sont des séquences synthétiques de peptide se liant à la protéine cible avec une affinité et une spécificité élevées. La première partie de ce travail porte sur l’étude électrochimique de l’interface modifiée résultant de diverses procédures d’immobilisation. Des mesures en présence du marqueur rédox [Ru(NH3)6]3+ ont démontré l’immobilisation de la sonde peptidique à la surface d’or et ont
permis l’évaluation relative de la densité de sondes adsorbées à la surface respectivement à la méthode d’immobilisation considérée. Par ailleurs, des mesures en présence du couple rédox [Fe(CN)6]3-/4- ont mis en évidence une inhibition drastique du transfert d’électron dans le cas
de monocouches composées exclusivement du peptide.
Dans un second temps, nous nous sommes intéressés à la détection de la protéine MDM2. Trois interfaces modifiées ont été envisagées soit deux monocouches mixtes de peptide et de 4-mercaptobutan-1-ol, ce dernier jouant le rôle de diluant, adsorbés en une ou en deux étape(s), et une monocouche uniquement composée de peptide. L’utilisation de la spectroscopie d’impédance électrochimique en présence du couple rédox [Fe(CN)6]3-/4- comme méthode de
détection a mis en exergue la pertinence de cette dernière interface pour la détection. En effet, l’inhibition du transfert d’électron préalablement identifiée est fortement amoindrie suite à l’interaction avec la protéine cible. Une gamme de détection s’étendant de ~1 à 60 ng mL-1 et
une limite de détection de 0,69 ng mL-1 ont été obtenues. Cette performance est comparable à
celle des kits ELISA commerciaux. La fiabilité et la spécificité de la réponse ont été vérifiées par le biais de contrôles négatifs sur trois protéines, en l’occurrence le fibrinogène, le cytochrome c et l’albumine de sérum bovin, et validées par des mesures complémentaires de microbalance à cristal de quartz.
La troisième partie de ce travail est consacrée à l’étude, par microscopie de fluorescence
in situ, de l’organisation de la monocouche résultant des trois procédures d’auto-assemblage
“This I believe: That the free, exploring mind of the individual human is the most valuable thing in the world. And this I would fight for: the freedom of the mind to take any direction it wishes, undirected.”
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Chapter 1. Introduction
1.1 Scope
The growing interest in point-of-care testing and the need of specific, reliable, fast, cost-effective and easy-to-use detection devices for clinical, biochemical and environmental analytes in various complex media has led to the development and elaboration of many types of sensors based on a wide range of probes such as antibodies, oligonucleotides, aptamers,… The interest in biological sensors is a very active area in analytical research [1-10]. Many of these biosensing related studies discuss the importance of an interdisciplinary approach involving a variety of fields such as biochemistry, material sciences, analytical chemistry…
A biosensor is a small device composed of a biological recognition element assembled on a transducer which transforms the biological event into a signal that can be directly measured. A variety of combinations of recognition elements and transducers can be found in the literature. Considering the increasing interest for biosensors, the International Union of Pure and Applied Chemistry (IUPAC) proposed the following definition [11]:
“An electrochemical biosensor is a self-contained integrated device, which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which
is retained in direct spatial contact with an electrochemical transduction element”.
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Figure 1-1: Schematic principle of a biosensor [2].
Biosensors can be classified according to the selected signal transduction method. Among these, electrochemical, optical and piezoelectric-based transduction are the most commonly considered. Electrochemical sensors, in which an electrode is used as the transduction element, indisputably attract the most interest due to some advantages: they are easy to design since no strict geometry, shape or size are required, they present low costs and are prospects to miniaturization. Up to now, developments have been performed in a wide range of applications such as clinical, environmental and agricultural analyses.
Biosensors can also be discriminated on the basis of the type of biorecognition element and the signal nature: enzymatic biosensors, based on the immobilisation of enzymes and, affinity biosensors, based on immunoreagents as antibodies or antigens, DNA derivatives as oligonucleotides sequences or aptamers and protein receptors.
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Besides antibodies, other proteins and peptides show the biorecognition properties required for the specific detection of biological targets. These alternative probes will be considered in this work.
The working principle of a biosensor rely on the selective interaction between an immobilised probe and a target protein. Electrochemical techniques allow the conversion of the formation of the duplex between a target in solution and the probe immobilised on a solid support (transducer) into an electric signal.
The conception of an electrochemical biosensor for the detection of biomolecules requires three key steps:
1. The immobilisation of the probe on the transducer surface 2. The biorecognition with the target protein
3. The electrochemical detection of the recognition Two approaches of electrochemical detection can be distinguished:
a labelled approach consisting in the modification of the probe or the target protein via the attachment of a redox centre aimed at providing a binding-sensitive electrochemical signal
a label-free approach in which no modifications of the target or the probe, aimed at providing an electrochemical response, are involved
Although, the biosensing strategies presented in this work will be classified according to these definitions, it should be underlined that there is no consensus about it, indeed, the modification of the probe is also often referred to as unlabelled.
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dramatically different results [35-37]. Besides, difficulties have been reported regarding their ability to achieve efficient interfacial architectures. The random orientation of these asymmetric molecules on surfaces can reduce their accessibility or even induce a loss of biological activity upon immobilisation [38, 39]. The rather big size (M>150 kDa) of these biomolecules can also cause some issues in terms of sensitivity. As biosensors are miniaturised devices, the use of smaller recognition elements could help overcome this problem.
Recently, a new biological tool, the peptide aptamer has emerged. Peptide aptamers, from the Latin aptus meaning “fitting”, consist of a short variable peptide domain presented in the context of a supporting protein scaffold [40]. Therefore, they resemble antibodies, in which a variable antigen-binding domain is exposed from a rigid backbone. Their ability to bind their target with high affinity and specificity both in vitro and in vivo makes them excellent candidates for protein analysis [41]. Conventionally, peptide aptamers are isolated from combinatorial expression libraries by screening systems based on the “yeast two-hybrid technology” developed by Stanley Fields et al. [42]. In the screening process, their coding sequence are isolated together with the aptamers, giving immediate availability and unlimited amount of the binding molecule. In an extended view, the word “aptamer” is also used for natural small peptides able to bind a specific target molecule. In the context of this work, we propose to work with this natural class of aptamers. More particularly, we propose to use a peptide based on the natural sequence of interaction of the p53 protein with the protein MDM2 as recognition element. Its in vitro synthesis will reduce the variability. Besides the smaller size of these biomolecules (< 35 kDa) which allows an improved sensitivity since a higher density of probe can be achieved, the modification of the peptide sequence with a cysteine residue at the N-terminal position should allow a better control of the orientation.
Regarding these considerations, we propose to contribute to the study of the immobilisation on a gold electrode of a peptide of the p53 tumour suppressor protein for the recognition of the oncoprotein MDM2.
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As a result, we will focus on three key axis, which are the immobilisation of probe, the electrochemical detection of the recognition event and the organisation of the biomolecules.
1.3 Immobilisation of the probe on the transducer surface
As mentioned earlier, the study of the molecular interactions occurring between two partners, the target contained in solution and the probe immobilised on the surface of a transducer, is often performed via the immobilisation of peptides and proteins on a surface transducer.
The key requirements for a sensor surface are:
1. Providing an optimal binding capacity of the probe
2. Retaining the biological activity of the probe since proteins tend to unfold when immobilised on a support [43]
3. Preserving the accessibility of the probe for the target 4. Minimising the non-specific adsorption
Obviously, surface chemistry is the initial key issue in the immobilisation of a protein or peptide probe [44, 45]. Contrary to negatively charged oligonucleotides, proteins are amphipathic molecules. Therefore, a high degree of adsorption, related to electrostatic and van der Waals forces, hydrophobic effect or conformational changes as well as a restricted lateral diffusion in the vicinity of the surface is observed. From one biomolecule to the other, the type and the extent of these interactions with the surface varies, complicating the achievement of zero-fouling surface.
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available functional groups, the surface must be specifically tailored to achieve the highest efficiency of covalent binding and optimise the homogeneity of the population of immobilised proteins.
Many types of organic reactions can be considered to covalently bind proteins to modified surfaces such as nucleophilic substitutions, esterification, acylation and nucleophilic addition methods [48].
Stine et al. recently reviewed on the various bioconjugation reactions for covalent coupling of proteins to gold surfaces [45]. The conjugation can occur through the amine group of the lysine residue side chain via its reaction with an activated modified surface as 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC or EDAC) and N-hydroxysuccinimide (NHS), or with SAMs terminated with aldehyde groups, or with epoxide functionalized surfaces.
Similarly, cysteine residues are also often used for immobilisation through their thiol side group. They can react with epoxides or conjugate addition to α, β-unsaturated carboxyl groups, such as maleimides to form thioester bonds. Figure 1-2 presents different ways of immobilisation via conjugation through nucleophilic residues.
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Figure 1-2: Various methods of immobilisation via conjugation through nucleophilic residues of proteins [45].
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Figure 1- 3: Mechanism for protein immobilisation via EDC/NHS coupling [45].
Wang et al. used binary mixed SAMs composed of protein resistant oligoethylene glycol thiol and N-hydroxysuccinimide terminated thiol for covalent attachment of proteins via lysine residues onto the surface and systematically studied isolated single molecules on surface through AFM measurements [52].
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Figure 1-4: DSP cross-linking approach for protein immobilisation on gold [45].
1.3.2 Direct immobilisation of the thiolated probe
Besides covalent coupling, it is also possible to directly covalently immobilise a peptide probe on gold. Indeed, a cysteine residue, containing a thiol group on its side chain, can be used as an anchoring group on gold.
The formation of self-assembled monolayers (SAMs) of thiolated molecules on gold surfaces is well known. Already in the 80s, Nuzzo et al. reported on the formation of organised layers of thiolated molecules on gold [54, 55]. These early studies focused on the immobilisation of three types of organosulfur compounds; alkanethiols, alkyl disulphides and dialkyl sulphides, with different alkyl chain lengths [56-60]. Since then, a massive amount of work has been dedicated to the understanding of the kinetics and thermodynamics associated with the organisation of these layers and many types of substrates (gold, palladium, silver, copper, platinum, carbon, mercury) and adsorbed molecules have been considered [61-69]. Although, alkanethiols monolayers on gold remain the most highly characterised.
Well-organised and compact self-assembled monolayers can be obtained via simple immersion of the substrate in a diluted solution containing the thiolated molecules. The high affinity of thiols for gold allows a fast adsorption, although it has been shown that many hours are required to obtain well organised layers [70, 71]. From these considerations, it is possible to immobilise molecules of biological interest containing a thiolated group through direct adsorption on gold. Therefore, the modification of a peptide probe by the addition of a cysteine residue in N-terminal position allows its direct immobilisation on gold.
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the sulphur-gold bonding, straighten up which favours the access for the target molecules. Figure 1-5 illustrates the effect of the passivation with an alkanethiol on a gold electrode previously modified with a peptide.
Figure 1-5: Illustration of the formation of a self-assembled monolayer by consecutive adsorption of a thiolated biomolecule and an alkanethiol.
Another common approach is the simultaneous adsorption of the biomolecule and the diluting thiol.
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1.4 Electrochemical detection of the recognition event
The analysis of proteins at electrode surfaces has been performed via various electrochemical methods. Among these, we can cite, for instance, the work of Brabec and Palecek investigating the presence of proteins at carbon electrodes, via differential pulse voltammetry or potentiometric stripping analysis [81-83]; the analysis relying on the oxidation peaks of tryptophan at about 0.70 V and/or tyrosine at 0.55 V vs Ag|AgCl reference electrode. However, no selectivity was endowed by the surface. Therefore, more complex interfaces, such as affinity-based sensors had to be considered.
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or a charge transfer resistance for impedance spectroscopy, is recorded in the absence of the target protein. Afterwards, upon interaction with the target protein, a “conformational change” induces a variation in the electron transfer rate between the redox group and the electrode, resulting in a modification of the electrochemical signal. Both positive (current increase) and negative (current decrease) variations have been reported. This probably arises from the fact that the monitored signal is a differential one and is therefore very sensitive to the structure of the peptide-modified electrode.
13 (A)
(B)
Figure 1-6: (A) Illustration of the labelled electrochemical peptide-based sensing approach developed by Gerasimov and Lai. (B) AC Voltammograms for the electrochemical peptide based sensor in the presence of various concentrations of anti-p24 antibodies and calibration curve showing the percentage of change in signal with increasing the target concentration [84,
85].
Two hypothesis explaining this change in the electron transfer have been formulated. While the first one assumed that the dynamics of the probe was modified upon binding, lowering the rate at which the redox label collides with the surface, the second explored the possibility of an envelopment of the label by the HIV-p24 antibodies, obstructing the electron transfer process.
14 (B)
Figure 1-7: (A) Illustration of the electrochemical peptide-based sensor for EGFR developed by Li et al. (B) Evolution of the current of the modified electrode incubated with increasing
concentrations of EGFR (10-10 g L-1 to 10-6 g L-1) [86].
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Figure 1-8: Illustration of the principle of the peptide-based sensor developed by Puiu et al. [88].
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binding of the target protein to the probe, a variation of the electrochemical signal is recorded. Among the different electrochemical methods which can be used to measure the modification of the electron transfer rate, impedance spectroscopy, from which the electron transfer resistance is easily extracted, is generally selected. Depending on how the binding affects the charge of the modified electrode and its accessibility, the rate constant can increase or decrease.
Estrela et al. achieved a label-free detection of protein interactions with peptide aptamers with and without addition of a redox marker [91]. They were able to monitor the protein detection both by open circuit potential measurements in absence of redox marker and, by following variations of the charge transfer resistance by means of electrochemical impedance spectroscopy in the presence of [Fe(CN)6]3-/4-. Figure 1-9 presents some of the data obtained.
They used peptide aptamers, recognising specific protein partners of the cyclin-dependent kinase (CDK) family, co-immobilised with PEG as a backfiller.
Figure 1-9: OCP measurements in 100 mM PB - pH 7.4(-150 to -200 mV vs Hg|Hg2SO4) and
EIS signal obtained in 100 mM PB - pH 7.2 in the presence of 10 mM [Fe(CN)6]3-/4- 1/1
(Frequency range: 10 kHz to 100 mHz; Edc= 195 mV vs Hg|Hg2SO4) for the detection of
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It can be seen on Figure 1-9 (left) that upon injection of rCDK2, the OCP follows a binding curve attributed to the interaction of the protein with the aptamer. Upon rinsing, non-specifically bound rCDK2 is removed from the surface and the OCP follows a dissociation curve reaching a stable value. Looking at the impedance data (right), an increase of 40 % of the charge transfer resistance is observed upon interaction. This rise is probably due to a combined effect of a larger amount of negative charges in the biolayer and further blocking of the surface hindering the redox process.
Miao et al. elaborated an electrochemical peptide-based sensor to evaluate apoptosis. The peptide probe was designed to contain the sequence recognising externalized phosphatidylserine (PS) on apoptotic cells and capture them on the surface of the gold substrate [92]. Indeed, upon apoptosis, PS translocates from the inner layer of the cell membrane to the outer layer. The selected peptide probe was based on the original sequence of the PS-binding site in PS decarboxylase and was co-immobilised with MCH as a backfiller. Figure 1-10 outlines the principle of the sensing mechanism for the apoptosis evaluation.
Figure 1-10: Illustration of the peptide-based approach for apoptosis evaluation developed by Miao et al. [92].
The incubation of the peptide-modified gold electrode in H2O2-treated cells allowed the capture
of apoptotic cells through specific recognition of the peptide and externalized PS on the cell membrane. Differential pulse voltammetry measurements were carried in the presence of the redox couple [Fe(CN)6]3-/4-. Before recognition, the positively charged peptide electrostatically
interacts with the negatively charged redox couple [Fe(CN)6]3-/4- and a large signal is recorded.
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Figure 1-11: (A) Differential pulse voltammograms for apoptosis evaluation. The top line corresponds to the peptide modified electrode whereas the other curves are recorded after incubated with cells previously treated with H2O2 (0, 25, 50, 80, 100 μM from top to bottom)
(B) Calibration plot of the modified electrode incubated with increasing concentrations of H2O2 [92].
Feng et al. prepared a label-free impedimetric sensor for the detection of cyclin A2 on non-covalent porphyrin functionalized graphene modified glassy carbon electrode [93]. The functionalization occurs through π- π stacking and hydrophobic interaction. Then, a specific hexapeptide binding to a surface pocket in cyclin A2 with high affinity is co-immobilised with a diluent, Tween 20, to prevent non-specific adsorption. Upon interaction with cyclin A2, the access to the electrode of the redox couple [Fe(CN)6]3-/4- in solution is hindered, increasing the
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Figure 1-12: Illustration of the impedimetric peptide-based sensing approach developed by Feng (up) and evolution of the electron transfer of the redox couple [Fe(CN)6]3-/4-for (a) bare
GCE; (b) TCPP/CCG modified GCE; (c) peptide linked to the TCPP/CCG modified GCE; (d) incubated in Tween 20 solution; (e) after addition 100 nM cyclin A2 protein on the modified
electrode (down) [93].
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Martic et al. proposed an “on chip” detection of sarcoma protein kinase and HIV-1 reverse transcriptase based on the immobilisation of two distinct peptides on gold [95]. They were able to simultaneously monitor two separate biochemical events using electrochemistry by both a label-free and a label-based approach. Their strategy is illustrated on Figure 1-14.
Figure 1-14: Illustration of the dual peptide-based sensor for the detection of sarcoma protein kinase and HIV 1 reverse transcriptase developed by Martic et al. [95].
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modulation of the current density of the immobilised ferrocene-labeled peptide upon binding. This strategy combines both a “signal on” and a “signal off” detection method.
It is interesting to note that, although label-free approaches have been widely used in DNA or antibodies-based sensors, very few work has been done, up to now, on peptide-based sensors compared to labelled approaches.
1.5 Organisation of the biomolecules monolayers
As already mentioned in this work, the stability of the layer and the accessibility of the probe are key issues in the elaboration of an efficient sensor. Therefore, the organisation of the monolayers resulting from the adsorption procedure has gained a lot of interest. In the context of DNA-based sensors, different groups tried to understand the influence of the immobilisation procedure on the organisation of the biomolecular probe at the electrode surface and more precisely on the homogeneity of the resulting self-assembled monolayer.
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Figure 1-15: Fluorescence image of a gold surface modified by MCH/ssDNA at open circuit potential (central) and illustrations of the origins of the variety of the
fluorescent features [99].
The evaluation of the DNA coverage was performed by reductively desorbing the fluorescent layer while simultaneously recording the capacitance and fluorescence evolution. These measurements showed that immobilising MCH prior to DNA leads to less densely DNA packed layers. They were able to show that the desorption process is influenced by the underlying substrate. The significant heterogeneities observed in the formation of DNA SAMs outlines the requirement of considering the influence of the procedure of immobilisation on the organisation of the monolayers when designing biomolecular sensors.
These heterogeneities and the organisation of DNA molecules on gold have also been studied via atomic force microscopy measurements by Josephs and Ye [101-103]. They observed subpopulations of probes with highly different levels of probe density and showed that the immobilisation procedure has an influence on the homogeneity of the SAMs. Indeed, monolayers prepared by inserting thiolated DNA into an alkanethiol monolayer were confirmed as being more homogeneous than those prepared through the passivation with an alkanethiol of a DNA preformed monolayer. However, it is hard to obtain a high density of probe through insertion of DNA because the process is limited by the defects in the preimmobilised MCH SAM.
23
measurements evidenced two successive signal increases which have been correlated to the substrate crystallinity confirming the observation made by the group of Bizzotto.
Although the major part of these studies focuses on DNA layers, some work has also been dedicated to the formation of peptide SAMs. While some interactions of the peptides with an electrode surface, including the ability of sulphur to covalently bind to various metal surfaces are of major interest since it allows the formation of stable self-assembled monolayers, others, commonly referred to as “fouling”, are often not desired. In many applications, as medical implants or biosensors, near-zero fouling monolayers present a high interest. In this context, many works have been devoted to the immobilisation of peptides allowing ultra-low fouling. For example, surface plasmon resonance (SPR) measurements realised by Bolduc et al. on 3-mercaptopropyl-amino acid monolayers have allowed them to characterise the tendency of serum proteins to non-specifically adsorb on gold according to the composition in amino acids of the immobilised peptide [51, 105, 106]. They showed that monolayers with polar and ionic amino acids with the shortest chain length were the most effective in reducing non-specific adsorption, regardless of the packing of the SAMs. Nowinski et al., also achieved a non-fouling and packed monolayer through the immobilisation of a peptide formed by alternating negatively charged glutamic acids and a positively charged lysine attached onto gold through an additional linker composed of four proline residues and a cysteine residue [107]. Chen et al. also focused their attention on the ability to form low-fouling peptide surfaces through the immobilisation of peptides based on glutamic acid, aspartic acid and lysine, either alternated or randomly mixed. They achieved a high resistance to non-specific protein adsorption (< 0.3 ng cm-2) comparable to that achieved by poly(ethylene glycol) (PEG)-based materials [108].
24
to form bicomponent peptide self-assembled monolayers. They showed that antiparallel conformation of peptide chains minimised the energy of interaction between the helix dipoles, allowing a stabilisation of the layer. This observation had already been reported by Fujita et al. [111], who showed that an antiparallel helix packing was significantly more favourable than a parallel one, suggesting that the SAM structure was regulated by dipolar interactions between helical peptides. STM and fluorescence measurements showed a rather homogeneous layer without one-component segregated regions.
Duchesne et al. designed a proximity probe based on chemical cross-linking to evaluate whether peptides are randomly distributed or if they self-reorganise to form supramolecular domains [112]. The probe was a peptide-capped gold nanoparticle covered with a binary peptide SAM composed of a matrix peptide and a longer functional peptide. Two reactive groups able to cross-link together or with a reactive group of another functional peptide were attached to each peptide. Depending on the proximity of other peptides, it will form an intramolecular bond or cross-link to another peptide. The inter vs intramolecular cross-linking allows the evaluation of the organisation of the peptide on the gold nanoparticle. They were able to show by comparing experimental results with a probabilistic model that peptides were not randomly distributed at the surface of the nanoparticle bur rather self-organised into supramolecular domains.
The effect of the length of the polypeptide on the assembled surface structure and of the molecular orientation within the self-assembled monolayer have been investigated by Sakurai et al. on Au (111) via AFM and FT-IR RAS measurements using three polypeptides PLLn-SH,
with n= 4,10, 30.They were able to show that α-helical PLL30-SH formed a homogeneous layer
with a 50° tilt angle towards the substrate whereas PLL4-SH and PLL10-SH formed
β-sheet-type SAMs arranged in small domains [76].
25
1.6 Strategy
1.6.1 Presentation of the considered system
1.6.1.1 The p53 tumour suppressor protein and the Murine Double Minute 2 oncoprotein
The p53 tumour suppressor protein is a transcriptional factor that plays a central role in the regulation of the cell cycle as it maintains the integrity of the cell by coordinating the cellular response to DNA damages via cycle arrest, DNA repair or apoptosis [113-116]. This 393 amino acids protein was identified in 1979 [117-119]. It can bind to specific DNA sequences and activate gene expression and this activity seems to be central to its function since tumour-derived mutants are defective in DNA binding. In 50 % of cancer, p53 is inactivated by mutations or other genomic alterations [120]. In other cancers, p53 is functionally inactivated by its primary cellular inhibitor, the murine double minute 2 protein (MDM2 or HDM2 in humans). Loss or malfunction of p53 is thought to contribute to the development of half of all cancers, including skin, breast and colon cancers.
MDM2 is an oncoprotein which was first evidenced by its overexpression in a spontaneously transformed mouse cell line [121, 122]. This ubiquitin E3 ligase binds to the N-terminal transactivation domain of p53 through its N-N-terminal domain. A ubiquitin E3 ligase is an enzyme involved in the conjugation of a ubiquitin protein to a substrate in order to promote its degradation by the proteasome. In tumours, gene amplifications and other alterations can result in elevated MDM2 and lead to the consecutive inhibition of p53. Amplification of MDM2 has been observed in more than one-third of soft tissue sarcomas and less often in glioblastomas, leukaemia, oesophageal carcinomas and breast carcinomas [123-129].
26
Figure 1-16: Illustration of the regulatory negative feedback loop of p53 and MDM2 [134].
Other proteins are also involved in the feedback loop as illustrated on Figure 1-16. The ARF protein (Alternate Reading Frame) binds to MDM2 and prevents degradation of p53 by inhibiting its ubiquitination. It is also know to inhibit MDM2 degradation. MDMX is a structural homolog of MDM2. It is able to increase both the levels of MDM2 and p53 through interaction with them. As homolog, it can compete with MDM2 for binding with p53. Besides, direct interaction between MDM2 and MDMX might interfere with the E3 activity of MDM2. The protein TSG 101 (Tumour Susceptibility Gene) interacts with both MDM2 and p53. It increase the half-time of MDM2 with consequent decrease of p53 level.
27
Figure 1-17: Structural features of p53 and human MDM2 (NLS: nuclear localized sequence, NES: nuclear export sequence) [134].
1.6.1.2 Formation of the MDM2-p53 complex
Many studies have been dedicated to the identification of the structure of MDM2 and p53 and to the understanding of the formation of the MDM2-p53 complex [135-144]. A combination of deletion and mutation analyses preformed on p53 and MDM2 allowed the determination of the approximate boundaries of the interacting regions of the two proteins[145-147]. A highly conserved (71 to 91 % across 5 species) 12 kDa structural domain, extending from residues 17 to 125, has been evidenced at the NH2-terminal portion of MDM2. This region
has been shown as necessary and sufficient to bind to p53 and is assumed to contain the p53 binding site. For p53, MDM2 binding has been evidenced as being dependent on a short linear sequence of 11 amino acids, extending from residues 17 to 26. This region is also highly conserved and is responsible for transactivation. Kussie et al. resolved the structure of the complex [137].
28
Figure 1-18: Diagram of the secondary structure elements constituting the MDM2 cleft [137].
Three amino acids of the p53 peptide are essential for the interaction. This invariant triad across species is composed of the hydrophobic and aromatic amino acids Phe19, Trp23 and Leu26 which insert into the MDM2 cleft. The interaction mainly relies on van der Waals and steric complementarity between the MDM2 cleft and the hydrophobic face of p53 helix since only two hydrogen bonds stabilise the complex. Figure 1-19 illustrates the p53-MDM2 complex.
29
1.6.1.3 Electrochemical sensing of MDM2
The negative regulator role played by MDM2 against the tumour suppressor protein p53 and the resulting loss of activity of p53 in case of overexpression of MDM2, makes MDM2 an interesting prognostic tool in many human tumours [148, 149]. MDM2 overexpression is supposed to increase the risk of distant metastasis, inducing a worse prognostic for the patient [124, 150, 151]. In many cell lines and in several tumour samples, it has been shown that overexpression of MDM2 can be correlated with a decreased response to both chemotherapy and radiotherapy. A simple explanation of the inhibition of therapeutic benefits of cytotoxic drugs and radiation has been found in the degradation role of MDM2 towards p53. Indeed, p53 is up-regulated by DNA-damaging agents, like chemotherapy and radiation, therefore the level of MDM2 increases as a result of its role in feedback control. The degradation of p53 increases, thus preventing cell cycle arrest and apoptosis.
Different detection strategies of the MDM2 protein have already been considered, such as immunohistochemistry which involves the labelling of proteins in a tissue sample with enzymes or with fluorescent tags, fluorescence in situ hybridisation on tumour tissue, using a fluorescent labelling of specific DNA sequences or chromosomes in a tissue sample to identify gene mutations or deletions [152-154]. More recently, the elaboration of electrochemical MDM2 sensors has been reported [155, 156].
The group of Zourob has elaborated a label-free impedimetric immunosensor for the detection of MDM2 in brain tissue [155]. The detection implies the formation of a cysteamine self-assembled monolayer on gold, further functionalised with MDM2 antibody using a homobifunctional 1,4-phenylene diisothiocyanate linker. The recognition event was followed by electrochemical impedance spectroscopy in the presence of [Fe(CN)6]3-/4-. Upon recognition,
30
Figure 1-20: Top: Immobilisation steps for the fabrication of the MDM2 immunosensor developed by Zourob et al. Bottom: Evolution of the charge transfer resistance as a function
of the concentration of MDM2 [155].
31
Figure 1-21: Illustration of the principle of the electrochemical immunosensor for the protein MDM2 developed by Li et al. [156].
32
Figure 1-22: Evolution of the current as a function of the concentration in MDM2 [156].
These two electrochemical approaches of detection of the protein MDM2 are the only reported to date. However, both methods are very complex and require many steps to achieve detection. In this work, we will present a simpler electrochemical procedure relying on the direct immobilisation of a peptide probe on a gold electrode. The selection of the sequence is based on the sequence of interaction of the protein p53 with MDM2. The recognition event will be detect by electrochemical impedance spectroscopy in the presence of the redox marker [Fe(CN)6]3-/4.
1.6.2 Choice of the electrode material
This study has been performed on a polycrystalline gold electrode. The ideally polarisable domain of the gold | aqueous solution system is limited by the reduction of the protons from the solution at negative potentials and by the oxidation of gold at positive potentials. Due to its wide electrochemical window, over 1 volt, gold is a model for the study of adsorption processes. Furthermore, obtaining reproducible surfaces free from impurities, an essential criterion in the study of these processes, can easily be achieved with gold as the stability of the electrode allows it to be flamed and/or electrochemically cleaned in solution [157, 158].
33
1.6.3 Selection of the sequence
The peptide aptamer sequence, selected for this study, is based on the transactivation domain of the p53 tumour suppressor protein and, is composed of the amino acids 12 to 26 containing the catalytic triad Phe19, Trp23 and Leu26 which, as presented earlier, are essential in the interaction with the oncoprotein MDM2. Schon et al. investigated the kinetics and thermodynamics of the p53-MDM2 interaction using a set of peptides based on residues 15-29 of p53 to understand the influence of the modification of the amino acids sequence on the binding [159]. They investigated the effect of the peptide length as well as natural and non-natural amino acid substitutions to define the specific amino acids residues required for the interaction.
They showed that p53(15-29) binds MDM2 with a dissociation constant of 580 nM. By modifying the 15-29 residue sequence they evidenced the influence of these changes on the binding efficiency. For instance, a 13-fold increased affinity was observed for the p53(17-26) sequence whereas further truncation to residues 19-26 supressed binding. Some explanation can be found in the mechanism of phosphorylation of the transactivation domain of p53 which is known to regulate the MDM2-p53 interaction. Indeed, phosphorylation at S15, T18 and S20 is thought to disrupt the interaction of p53 with MDM2. Therefore they evaluated the impact of phosphorylation of T18, a highly conserved residue particularly in mammalian p53 and evidenced a weakened binding to Mdm2 by about an order of magnitude. Although this residue does not directly interact with MDM2, its substitution showed its importance for binding. As a matter of fact a radical change in the peptide-binding behaviour was monitored when T18 was deleted. Truncation of T18 completely abolished the binding. From these observation, Schon et al., reached the conclusion that truncation at residue T18 might represent the minimal peptide length for MDM2 binding. As a matter of fact, the minimal length for a tight-binding peptide was reached with p53(18-26) that presented a dissociation constant of 70 nM. Furthermore, following Kussie et al., T18 is involved in the formation of an intramolecular hydrogen bond with D21 by stabilizing it in the α-helical conformation [137]. The importance of this residue is supported by the fact that phosphorylation of S15 and S20 did not affect binding.
They also showed that shorter p53-derived peptides present tighter binding. The peptide p53(17-26) binds MDM2 ten times more tightly (Kd=46 nM) than the wild type p53(15-29)
(580 nM).
34
relying on the immobilisation of an aptamer of the protein p53 on gold. This method is based on electrochemical impedance spectroscopy measurements in the presence of the redox couple [Fe(CN)6]3-/4-. Negative controls will be considered in order to assess the selectivity and
specificity of the sensor whereas quartz crystal microbalance measurements will be used to validate the electrochemical signal as originating from the interaction between the target and the probe.
35
Chapter 2. Experimental
2.1 Electrochemical techniques
2.1.1 The electrochemical cell
Electrochemical experiments are performed in a three-electrode cell at room temperature. Data are recorded using an Autolab PGSTAT30 (Eco Chemie, The Netherlands) potentiostat controlled by GPES 4.9, FRA 4.9 or NOVA software.
Prior to every measurement, supporting electrolytes are purged with nitrogen for at least 15 minutes to remove any trace of oxygen which can easily be reduced under polarization and might interfere with electrochemical measurements. During measurements, the solutions are kept under nitrogen to prevent dissolution of oxygen from the atmosphere.
36
Figure 2-1: Representation of the three-electrode cell used for the electrochemical measurements.
The working electrode are polycrystalline gold disks with a 1.6 mm diameter from BASI (Bioanalytical Systems, USA). As adsorption processes at the electrode|solution interface depend both on the electrode nature and on the surface cleanliness, it is of main importance to characterise the electrode before any subsequent manipulation. Prior to every measurement set, the polycrystalline gold electrode surface was polished with 1.0 µm alumina-water slurry on a smooth polishing cloth (Struers), sonicated for 2 minutes and rinsed thoroughly with Milli-Q water. Then, the electrode was electrochemically cleaned by cycling the potential between -0.3 V and +1.5 V in 0.1 M HClO4 solution at a scan rate of 50 mV.s-1 until
37 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 -14 -12 -10 -8 -6 -4 -2 0 2 4 i / µA E vs Ag|AgCl / V
Figure 2-2 : Cyclic voltammograms of a polycrystalline gold electrode in 0.1 M HClO4
starting from -0.3 V until +1.40 V ( ) and +1.50 V ( ); υ=50 mV s-1.
On the forward scan, while sweeping to more positive potentials, the oxidation of the gold electrode to the state Au (III) results in an intense peak around +1.1 V followed by a lower intensity. The reduction of the gold oxides occurs on the backward scan and is evidenced by an intense peak at +0.9 V. Comparing the experimental voltammograms to those found in literature witnesses the quality of the substrate [157, 158].
The roughness of the electrode has been taken into account by normalising every electrochemical data to the real electrode area. It has been estimated based on the gold voltammograms recorded to +1.4 V, potential at which a monolayer of gold oxide is assumed to be formed. The cathodic charge associated with the reduction of a monolayer of oxide is estimated at 400 µC cm-2 [160]. After characterisation, the gold electrode was rinsed with Milli-Q water and dried under a nitrogen stream.
38
as a function of time. Consecutive scans can be performed and the resulting current is plotted as a function of the applied potential.
Figure 2-3: Profile of the applied potential as a function of time in cyclic voltammetry.
In cyclic voltammetry, the measured current is always the sum of two contributions of different nature. The first is the faradaic current, IF, originating from oxidation and reduction reactions, which follows Faraday’s law. The second is the capacitive current, IC, related to the presence of an electric double layer at the electrode|solution interface.
39
where I is the total current measured, QF the faradaic charge, t the time, C the double layer capacitance assumed to be constant and υ the scan rate.
The scan rate is an important parameter in cyclic voltammetry. Indeed, equation 2.3 indicates that the capacitive current is proportional to the scan rate. Besides, the faradaic current also depends on this parameter.
For a redox couple exhibiting fast electron transfer kinetics (“reversible”) in solution and under a semi-infinite linear diffusion of the electroactive species to the electrode surface, the peak current Ip at 25 °C is given by:
𝐼𝑝 = 2.69 × 105𝑛3/2𝐴 𝐷1/2𝑐∞𝜐1/2 (2.4)
with Ip the peak current (A), n the number of electron transferred, A the area of the electrode (cm2), D the diffusion coefficient of the electroactive species (cm2 s-1), c∞ its bulk concentration (mol cm-3) and υ the scan rate (V s-1).
Figure 2-4 presents a cyclic voltammogram for a reversible process O + e- R when initially
only O is present in solution.
Figure 2-4: Cyclic voltammogram for a reversible process O + e- R when initially only O is present in solution [161].
40
In the case of a reversible process where the electroactive species are adsorbed at the electrode, the peak current is given by:
𝐼𝑝 =𝑛
2𝐹2
4𝑅𝑇 𝐴 Γ 𝜐 (2.7)
where Γ is the surface concentration of adsorbed species.
Figure 2-5 presents a cyclic voltammogram for reduction and subsequent reoxidation of an adsorbed species O.
Figure 2-5 : Cyclic voltammogram for reduction and subsequent reoxidation of an adsorbed species O. Current is given in normalized form and the potential axis is shown for 25°C [162].
For an irreversible process, the peak current observed for adsorbed electroactive species is given by:
𝐼𝑝 =
𝛼𝐹2
41
42
present work, it offers the advantage of a better separation of the contributions, originating from the electric double layer and, the faradaic responses involving adsorbed or diffusing species. Figure 2-6 presents the profile of the imposed potential jump (a) and the associated responses in current (b) and in charge (c).
Figure 2-6: (a) Potential progamming in chronoamperometry; (b) corresponding current transient curve; (c) charge transient curve resulting from the integration of the current
transient.
43
2.1.4 Electrochemical Impedance Spectroscopy
The electrochemical techniques described previously, impose relatively large perturbations to the system, either via a potential sweep or a potential step, to study the reactions occurring at the electrode surface.
Electrochemical Impedance Spectroscopy is based on a different approach. An alternating potential (ac) of small amplitude, Eac, is superimposed to the dc potential, E, applied at the working electrode. The resulting alternating current is then followed, at a fixed potential E, as a function of the frequency of the ac perturbation [163].
The alternating potential is expressed by:
𝑒𝑎𝑐 = 𝐸𝑎𝑐sin (𝜔𝑡) (2.9)
The alternating current is given by:
𝑖𝑎𝑐 = 𝐼𝑎𝑐sin (𝜔𝑡 + 𝜙) (2.10)
where ω=2πf is the angular frequency, f is the frequency expressed in Hz and Φ is the phase angle.
The alternating current and potential can be represented as separated phasors, 𝐸̇ and 𝐼̇, rotating at the same frequency as shown on Figure 2-7.
44
where Z is the impedance of the system which can be represented in the complex notation by:
𝑍 = 𝑍′− 𝑖𝑍′′ (2.12)
where Z’ and Z’’ are the real and imaginary parts of the impedance. The magnitude of the impedance, |Z| is given by:
|𝑍|2 = (𝑍′)2+ (𝑍′′)2 (2.13)
and the phase angle Φ is given by:
𝜙 = 𝑡𝑎𝑛−1(𝑍′′
𝑍′) (2.14)
The variation of the impedance of a system as a function of the perturbation frequency can be represented in different ways. The most commonly used representation is the Nyquist plot displaying Z’ as a function of -Z’’ for different frequencies. Another frequently used representation is the Bode plot presenting both log|Z| and Φ as a function of log f.
Equivalent circuits are used to model electrochemical systems in order to extract a maximum of information on the electrode processes. They are composed of a combination of resistances and capacitors or other components allowing to explain the observed phenomena (Warburg impedance, Constant Phase Element, inductance,…).
45
Figure 2-8: Randles equivalent circuit.
46
moment. The application of a periodic perturbation (oscillating electric field), to the edge of the solid, results in an oscillating elastic deformation. The latter travels through the solid as a transversal acoustic wave, which is reflected at its ends.
The quartz is a crystal presenting both mechanical, electrical, chemical and thermal properties allowing a mechanical deformation upon electrical stress. The wave propagation mode depends on the cut of the crystal. Although a variety of crystals are suitable for quartz crystal microbalance measurements, AT-cut are the most commonly used. They present a cut angle of 35.25 ° to the z-axis with the advantage that, between 0 and 50 °C, they exhibit a negligible frequency drift as a function of temperature. As a result, this cut is well-suited for QCM sensing. When the wavelength of the acoustic wave is twice the thickness of the quartz crystal, the eigenfrequency is reached. In these conditions, the resonance frequency of the acoustic wave can be expressed by equation 2.15:
𝑓0 = 𝜐𝑄
2𝑡𝑄 (2.15)
with f0 the resonance frequency, υQ the velocity of the sound in the crystal (3.34 × 104 m s-1 for an AT cut quartz crystal) and tQ the thickness of the crystal.
Upon deposition of a layer of material on top of the crystal, the path of the acoustic wave is modified and, accordingly the resonance frequency. This variation of the resonance frequency can be related to the variation in mass via the Sauerbrey equation assuming a few hypotheses regarding the characteristics of the deposited layer. The validity of this equation is restricted to thin uniform rigid layers presenting a density similar to that of the crystal. According to these assumptions we have:
∆𝑓 =−2𝑓0
2 ∆𝑚
𝐴√µ𝑄𝜌𝑄
= −𝑆∆𝑚 (2.16)
where Δf is the variation of the frequency, f0 the resonance frequency, Δm the mass variation, A the electrode area (cm2), µ
47
For QCM measurements, only one side of the gold coated crystal was modified with the peptide layer and put in contact with the buffer. A gold crystal with a resonance frequency of 10 MHz and an area of 0.205 cm2 was used. Microbalance measurements were performed on a Gamry instrument with the flow cell as shown on Figure 2-9. Electrochemical coupling allowed the characterisation of the crystal surface prior to immobilisation.
(a) (b) (c)
48
Figure 2-10 : Schematic principle of AFM.
There are various mode of imaging in AFM, among which three are the most common:
1) The contact mode where the tip remains in contact with the sample and the deflection of the cantilever is recorded based on repulsive forces.
2) The tapping mode in which the cantilever oscillates at its resonance frequency. When there is a topographic change at the surface, the oscillation is disturbed and the piezoelectric needs to adapt to maintain the oscillation amplitude constant. The movement of the piezoelectric as a function of x,y gives the sample topography. 3) The non-contact mode where the probe is not in contact with the sample but oscillates
above the surface based on the attractive forces with the sample.
The image resolution depends on the interaction between the tip and the sample but also on the tip size and the tip-to-sample distance. The image is then constituted of a series of parallel lines composed of pixels characterised by their position (x,y) and height (z).
49
2.4 In situ Fluorescence Microscopy under polarisation
The technique of in situ fluorescence under polarisation, coupling electrochemistry with fluorescence constitutes a major part of this work. We decided to develop the theoretical considerations latter in this manuscript, along with the presentation and discussion of experimental results. In this section, we will focus only on the equipment and on the description of the coupling between the potentiostat and camera.
2.4.1 Experimental setup
Every fluorescence measurement was performed in a specially designed and manufactured electrochemical cell, shown on Figure 2-11. This cell was obtained thanks to the courtesy of Professor Bizzotto and built by Brian Ditchburn, the glassblower of the Chemistry Department of the University of British Columbia, Vancouver, Canada.
Figure 2-11: Representation of the three-electrode cell used for in situ epifluorescence microscopy.
50
characterised by cycling the potential between -0.3 V and +1.5 V in 0.1 M HClO4 until
reproducible cycles were obtained to ensure the quality of the substrate. For in situ fluorescence microscopy measurements under potential control, the gold bead, connected to a copper wire sealed in a glass tube, was placed in contact with the electrolyte solution using the hanging meniscus technique. Only the flat surface of interest of the electrode was thus in contact with the electrolyte. This specific configuration of the cell is adapted to the use of an inverted microscope in which the sample is analysed from the bottom rather than from the top, looking up at the surface of the electrode. The microscope is connected to a CCD digital camera allowing the recording of images. The coupling between electrochemistry and fluorescence, which will be described in the following section, is operated via triggering.
51
Table 2-1 : Description of the equipment used for in situ fluorescence measurements.
UBC ULB
Microscope Olympus IX70 Eclipse Ti, Nikon
Lamp EXFO X-Cite eXacte
Mercury Lamp
HBO 103W/2 Mercury Lamp
Objective 10 × Olympus LM PlanFl (NA 0.25; WD 21 mm)
Nikon CFI Plan Fluor DIC L (NA 0.30; WD 16 mm)
Objective 50 × Olympus LM Plan-Fl
(NA 0.5 ; WD10.6 mm)
Nikon CFI LU Plan EPI ELWD
(NA 0.55 ; WD 10.1 mm) Fluorescence Filter Cube WIBA cube
Excitation: 450-490 Dichroic mirror: 505 Emission: 510-550 Nikon B2A Excitation:450-490 Dichroic mirror: 505 Emission: 520 LP
CCD Camera EvolveTM 512 EMCCD
Camera, Photometrics Clara Interline CCD Camera, model DR-328G-C01-SIL, Andor Technology
Softwares Labview program Andor Solis Software +
NOVA
The numerical aperture (NA) describes the acceptance cone of an objective and hence, its ability to collect light. Usually, bigger NA are preferred because resolution is inversely proportional to the numerical aperture as expressed in equation 2.17.
𝑅 = 𝜆
2𝑁𝐴 (2.17)
where R is the resolution, λ is the illuminating wavelength and NA numerical aperture.
52
Objective 10 × 0.647 pixels/µm 1.7 pixels/µm
Objective 50 × 3.235 pixels/µm 8.7 pixels/µm
2.4.2 Electrochemical coupling
In this technique, changes in potential, electrochemical responses, and fluorescence images were triggered and recorded simultaneously. Two distinct triggering have been used with the two equipments.
For the measurements performed at the University of British Columbia, electrochemical measurements were performed on a (HEKA PG590 with PAR 175 scan generator) and lock-in amplifier (EG&G 5210). The differential capacitance was measured using a 200 Hz, 5 mV RMS ac perturbation superimposed onto the dc potential. The resulting current response was analyzed by a lock-in amplifier. The in-phase and out-of-phase amplitudes were then used to calculate the capacitance assuming a series RC circuit. The triggering of the potentiostat followed by that of the camera is controlled by a custom Labview program. Once the images have been taken by the camera, it sends back a feedback (trigger out) to Labview which can proceed to the next step.
Measurements performed at the Université libre de Bruxelles were performed on a µAutolab equipped with a Frequency Response Analysis module using NOVA 1.7 software. The differential capacitance was measured using a 183 Hz, 5 mV perturbation. The capacitance is calculated assuming a series RC circuit. When the desired potential is applied to the electrode, the NOVA software triggers the camera which takes an image. The trigger is then turned off and the impedance measurement is performed.
53
Figure 2-12 : Illustration of the potential programming and associate triggering used during in
