Interfacial study of a sensing platform for MDM2, based on ... · Léo, j’ai toujours pu compter...

UNIVERSITÉ LIBRE DE BRUXELLES

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

Supervisor: Pr. Claudine Buess-Herman

Co-supervisor: Dr. Thomas Doneux

September 2015

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.

Ma thèse ayant été étroitement liée à l’enseignement des travaux pratiques de chimie

analytique je tiens à remercier l’ensemble des étudiants que j’ai côtoyés au cours de ces six

années. Cette expérience a été pour moi une immense source de satisfaction et

d’enrichissement personnel! Ces journées l’auraient paru beaucoup plus longues sans mes

collègues de TP que je remercie vivement : Denis, Jennifer, Clément, Steph C. et Yannick.

En parlant de TP, il y a une personne qui m’a énormément apporté… Eric, voilà six années

je débarquais comme jeune assistante, peu sûre de moi, dans l’envers du décor des labos. Grâce

à toi, je me suis adaptée rapidement et m’y suis sentie comme à la maison. Les petits déjeuners,

les chocolats, les morceaux de tarte autour d’une tasse de café, nos longues discussions, tous

ces moments partagés ont fait de toi, non plus un collègue, mais un ami. Tu as été un soutien

sans faille dans les moments plus difficiles et je ne t’en remercierai jamais assez.

Les filles, tout un programme, qu’est-ce qu’on a ri! Vous m’avez toujours soutenue et

comprise. Perrine, Julie, Steph et Dédé, j’ai beaucoup de chance de vous avoir rencontré et j’ai

hâte que nous continuions à partager de nouvelles aventures! Les boys, Titi et Sami, que dire si

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.

Julius, tu as dû faire preuve de beaucoup de patience depuis que nous nous sommes

rencontrés. Je n’ai pas toujours été disponible mais tu t’es montré très compréhensif, mieux tu

m’as encouragé à persévérer dans mes ambitions. Tu arrives à me faire sortir de ma zone de

confort et tu fais de moi une personne meilleure. Tu m’acceptes dans toute ma complexité.

Merci d’être là.

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

2.4.2 Electrochemical coupling ........................................................................................... 52

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 Reductive desorption behaviour of mixed layers …………….....................................118

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

6.2 Prospects ………………………..……….....................................................................141

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.

The third part of this work is dedicated to the in situ fluorescence microscopy study of the

organisation of monolayers resulting from the three pre-cited immobilisation procedures. These

measurements evidenced a heterogeneous distribution of the probe density and more

particularly the presence of aggregates. These features cannot be desorbed from the electrode

surface via very negative potentials (-1.450 V vs Ag|AgCl). The influence of different

parameters such as the structure of the electrode, the presence of urea or the absence of an

anchoring function on the probe have been studied. Finally, atomic force microscopy

measurements have completed this study.

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

précitées. Ces mesures ont permis la mise en évidence d’une distribution hétérogène de la

densité en sondes, et plus particulièrement la présence d’agrégats. Ceux-ci ne peuvent être

désorbés de la surface par l’application de potentiels même très négatifs (-1,450 V vs Ag|AgCl).

L’influence des différents paramètres tels que l’état de surface, la présence d’urée ou l’absence

de fonction d’ancrage sur la sonde a été étudiée. Enfin des mesures de microscopie de force

atomique ont complété cette étude.

“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.”

John Steinbeck

Chapter1 Introduction

1

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”.

Although this definition focuses on electrochemical biosensing, it can be extended to other

principles of signal transduction. Figure 1-1 presents a typical scheme of a biosensor.


2

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.

In the case of enzymatic sensors, the signal reflects the rate of the substrate conversion or

enzymatic activity. For example, electrochemistry of immobilised proteins has been used for

the detection of electroactive species. Cytochrome c modified electrodes have been used for the

analysis of superoxide as well as haemoglobin modified electrodes for nitric oxide detection

[12-16] Metal binding properties of proteins have also been used for metal ion analysis [17-20].

Affinity-based biosensors, on the other hand, exploit reversible biochemical interactions as

antigen-antibody, DNA-DNA or DNA-protein [7]. Among these, immunosensors based on

antibodies or antigen as bioreceptor are the most widespread. The high affinity and specificity

of an antibody for its antigen allows a selective binding of the analyte in the nano- to picomolar

range in the presence of hundreds of other substances, even if they exceed the analyte

concentration by 2-3 orders of magnitude. Furthermore, antibodies present a wide versatility in

nature and are commercially available. Their use as recognition elements in bioanalytical assays

can be traced back to the late 1950s [21]. Since then, a lot of work has been devoted to the

development and understanding of these devices [22-29]. Nowadays, the most popular

immunosensor is the enzyme-linked immunosorbent assay (ELISA) allowing the detection of

an antibody or antigen in a complex sample.


3

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.

Each of these three steps has to be optimised to guarantee a high-performance sensor. A wide

variety of substrates as carbon, mercury, platinum, gold and conducting polymers can be used

[30-34].


4

1.2 Aim of the work

In the context of this thesis, we will focus on the elaboration of an electrochemical affinity-

based biosensor and more particularly on the immobilisation of peptides for the electrochemical

detection of proteins. Indeed, although antibodies are one of the key tools used in biological

sciences for the identification of other molecules, it is known that they presents quite a number

of drawbacks. Among these, there is the batch-to-batch variability which can produce

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.

As the stability of the layer and the accessibility of the probe are key issues in the

elaboration of an efficient sensor, electrochemical detection of biomolecules requires a deep

understanding of the surface chemistry. Therefore, this study comes within the wider scope of

the elaboration and comprehension of molecular self-assembly. The study of the organisation

of the biomolecular probe at the electrode surface resulting from the adsorption procedure and

more precisely of the homogeneity the self-assembled monolayers is of major importance.


5

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.

Although physical adsorption represents the easiest process for probe immobilisation, it is

not suitable in the context of biosensing since it is almost impossible to control. Indeed, a close

proximity of the recognition site to the surface can suppress the affinity for the target.

Furthermore, in the presence of a mixture of proteins in solutions, the adsorbed proteins may

exchange with those present in solution whereas stringent washing can destabilise the protein

attachment [46, 47]. This indicates that none of the key requirements presented above is

fulfilled. Therefore, in the context of this work, we will consider a covalent binding of the

probe.


6

1.3.1 Covalent coupling

The immobilisation of proteins, such as antibodies, on a surface is often performed through

covalent binding with reactive groups on the support. As peptides present similar chemical

functionalities as proteins, for example, -SH (cysteine), -NH2 (lysine, arginine), -COOH

(asparagine, glutamine), -OH (serine), imidazole (histidine) on the side chains of their

backbone, it is possible to immobilise them using the chemistry developed for antibodies such

as chemical coupling reactions with prepared SAMs. To take advantage of the naturally

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.

Fernandez-Lafuerte et al. proposed a way of immobilisation based on the activation of the

carboxyl groups of aspartic acid and glutamic acid by reaction with a carbodiimide [49].


7

Figure 1-2: Various methods of immobilisation via conjugation through nucleophilic

residues of proteins [45].

The EDC/NHS cross linking approach is one of the most commonly used. EDC is a zero-

length cross-linking agent used to couple carboxyl groups to primary amines. This approach

requires a preliminary functionalization of the gold substrate by a sulphur containing species

with a reactive terminal functional group as mercaptopropionic acid or an alkanethiol [50, 51].

The modified electrode can then react with EDC to form an amine-reactive intermediate, an O-

acylisourea. This intermediate is unstable and requires stabilisation via N-hydroxysuccinimide

to form an amine reactive NHS-ester. Subsequently, in the presence of the protein,

immobilisation occurs through displacement of NHS groups by lysine residues of the protein.

Figure 1-3 presents the chemical reactions associated with the EDC/NHS coupling.


8

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].

Suri et al., also used a cross linking approach as they modified gold surfaces using a

homobifunctionnal cross-linker, the dithiobissuccinimide proprionate (DSP) for protein A

immobilisation [53]. Indeed, protein A can then play the role of a linker to immobilise

antibodies in a uniform, stable and accessible way. Figure 1-4 presents this cross-linking

approach.


9

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.

The immobilisation of peptide or protein probes, via the thiol side chain of a cysteine

residue, has already been studied and is known as a suitable way to self-assemble peptide

sequences on gold [72-76]. It is known that peptides and proteins, as it is the case for DNA,

also tend to non-specifically adsorb on gold and this can significantly reduce the performance

of a sensor.


10

To circumvent non-specific adsorption, biomolecules are often coadsorbed with small

alkanethiols as 4-mercaptobutan-1-ol or 6-mercaptohexan-1-ol playing the role of diluent.

Various ways of direct immobilisation of biomolecules in the presence of a diluent on gold are

considered in the literature. Among these, a two-step adsorption, proposed by Tarlov et al.,

consisting of the immobilisation of the biomolecule probes, followed by the passivation of the

surface with an alkanethiol diluent is commonly used [77, 78]. This type of immobilisation

allows a better accessibility of the probe and prevents non-specific adsorption as it has been

shown with oligonucleotides SAMs. Indeed, the molecules, only covalently anchored through

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.

Electrochemical measurements, carried out on oligonucleotides directly immobilised on

gold, have shown that the ionic strength of the immobilisation solution has a significant

influence on the amount of DNA immobilised on the surface [79, 80]. As DNA which is

negatively charged, peptides also carry charges, therefore, electrostatic repulsions between the

strands tend to decrease the amount of immobilised biomolecules. This effect can be circumvent

using a high ionic strength solution for the immobilisation step. Accordingly, high ionic

strength solutions have been used to prevent charge effects during immobilisation and achieve

densely packed layers.


11

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.

The selectivity of an electrode for a target protein is based on the immobilisation of a

specific ligand at the surface, acting as biorecognition element. Among these, we mostly find

antibodies but we mentioned earlier a new class of ligands, called aptamers, composed of

synthetic single-stranded nucleic acids or peptides presenting a high affinity for proteins has

emerged. Peptide ligands were initially developed as research tools to dissect protein function

within complex molecular regulatory networks [41, 40]. Their ability to bind the target protein

with high affinity and specificity, and their smaller size compared to antibodies makes them

powerful alternatives for the detection of proteins. Besides a good selectivity, a sensor has to

offer a high sensitivity. In this context, electrochemical methods are often considered.

Therefore, we will focus on the detection of biomolecules through the elaboration of

electrochemical peptide-based sensors. As mentioned previously, two approaches of

electrochemical detection can be considered, a label-based detection and a label-free detection.

In the two following sections, we will present a state of the art of the studies dealing with label-

based and label-free electrochemical peptide-based sensors.


12

1.4.1 Label-based electrochemical detection

The label-based detection, which is the most widely used, consists 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. The covalent linkage of a redox group to the probe extremity

is one of the common transduction means considered.

Practically, the electrochemical response of the probe-modified electrode, which can be a

current, as in cyclic voltammetry, differential pulse voltammetry or square wave voltammetry,

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.

Gerasimov and Lai developed an electrochemical peptide-based sensing platform for the

detection of HIV anti-p24 antibodies based on the use of methylene blue (MB) as a reporter of

the interaction [84, 85]. They immobilised an antigenic epitope from the HIV-1 capsid protein,

p24, with the sequence EAAEWDRVHP, modified at the N-terminus with an 11-carbon thiol

to allow the anchoring on the gold substrate and at the C-terminus with an MB redox label via

the side chain of an added lysine residue. The redox signal was measured by AC voltammetry.

Upon binding with the target, a suppression of the electron transfer rate to the methylene blue

reporter is observed. Figure 1-6 illustrates this approach.


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.

Li R. et al. also developed a peptide-based sensor, as shown on Figure 1-7, relying on the

electron transfer from a ferrocene (Fc) moiety attached on the peptide probe for the detection

of epidermal growth factor receptor (EGFR) [86]. The immobilised 12-mer Fc-modified ligand

shows a voltammetric signal due to the one-step reduction of the ferrocenyl group. Upon

recognition with EGFR, an enhanced signal is detected. The amplitude of the current increases

and presents a linear relationship with the logarithm of EGFR concentration. Li H. et al. used a

similar approach for the detection of amyloid 1-42 soluble oligomers although a “signal off”

was recorded [87].


14

(A)

(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].

Puiu et al. reported on a modular electrochemical peptide-based sensor for the detection of

anti-deamidated gliadin peptide (DGP) antibody [88]. The immobilisation of a short helical

support peptide on the surface of a gold electrode is followed by its labelling with a methylene

blue redox marker. The tagged support peptide is further modified with a recognition peptide,

an alpha-2 deamidated gliadin peptide (DGP), a 33-mer peptide containing the residues 56-88

of alpha gliadin from gluten. It has been shown that relatively low surface densities of support

peptide allowed the achievement of a highest labelling yield. The recognition of the target, the

anti-DGP IgG monoclonal antibody, leads to a signal decrease. Indeed, the electrochemical

signal recorded in the absence of the target antibody is due to electron transfer through the

support peptide and through a collisional mechanism due to the relative flexibility of the support

peptide that allows the MB to collide to the surface and transfer electrons with good efficiency

[89]. Upon recognition the flexibility of the anchored entity and the electron transfer are

modified, suppressing the signal. Figure 1-8 presents this strategy.


15

Figure 1-8: Illustration of the principle of the peptide-based sensor developed by Puiu et al.

[88].

Sugawara et al. based their sensor for ovalbumin detection on a peptide involved in the

lysozyme [90]. They immobilised, on a glassy carbon electrode, the peptide corresponding on

the amino acids 112 to 123 on the C-terminal side of lysozyme which selectively combines with

ovalbumin. An electroactive label, the daunomycin, was introduced as reporter at the N-

terminal side of the peptide, using a cross-linking reagent, the ethylene

glycobis(sulfosuccinimidyl)succinate. Using the oxidation wave of daunomycin, they observed

a decrease in the electrode response as the concentration of ovalbumin increased.


16

1.4.2 Label-free electrochemical detection

In a label-free detection method, no modifications of the target or the probe, aimed at

providing an electrochemical response, are involved. However, with the notable exception of

double layer capacitance measurements, the addition of a redox active molecules is needed.

The most popular redox marker is the ferri/ferrocyanide redox couple [Fe(CN)6]3-/4- [26-28] .

The apparent electron transfer rate of the redox couple is strongly dependent on the accessibility

of the redox molecule to the electrode surface. Therefore, upon modification of the interface by

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

rCDK2 based on the elaboration of a peptide aptamer based sensor [91].


17

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.

Once the apoptotic cells have been captured, the positive charges of the peptide can be hidden


18

and a drop of the electrochemical signal is observed. Experimental results indicated that a

higher level of H2O2 induced a higher number of apoptotic cells. Figure 1-11 presents the results

obtained.

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

charge transfer resistance measured by electrochemical impedance spectroscopy as shown on

Figure 1-12.


19

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].

Kerman et al. elaborated an electrochemical peptide-based sensor for the measurement of

the activity of protein kinase [94]. They immobilised a peptide probe specific for CDK2 via a

lipoic acid-succinimide ester and passivated the surface with PEG to prevent from non-specific

adsorption of protein on the sensor. Adenosine 5’-γ-[ferrocene triphosphate] (Fc-ATP) was

used as a reporter, since it enables the kinase-catalysed transfer of a redox active γ-phosphate-

Fc to a hydroxyamino acid of the immobilised peptide. Immersing the sensor in a solution

containing CDK2 and Fc-ATP into HeLa cell lysates, they managed to follow the

phosphorylation of the immobilised peptide using square wave voltammetry of the surface-

confined Fc molecules. Figure 1-13 illustrates this strategy.


20

Figure 1-13: Illustration of the peptide-based sensing approach developed by Kerman to

evaluate the activity of protein kinase [94].

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].

Indeed, the detection of the sarcoma protein kinase is based on the same approach as that used

by Kerman et al. using ferrocene-labelled adenosine triphosphate (Fc-ATP) that has been

presented above whereas the detection of HIV 1 reverse transcriptase protein is based on the


21

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.

The group of Bizzotto, from the University of British Columbia, studied the organisation

of self-assembled monolayers of various molecules such as alkanethiols, lipids, or DNA

strands, using in situ epifluorescence microscopy under potential control [96-100]. In their work

on DNA, they compared two procedures of immobilisation of mixed SAMs of mercaptohexanol

and fluorescently labelled DNA. The first immobilisation procedure was based on the

traditional Tarlov method where the DNA modified electrode is passivated by MCH

(DNA/MCH SAM). The second immobilisation procedure consisted of the immobilisation of

MCH followed by DNA (MCH/DNA SAM) [99]. Fluorescence images recorded at open circuit

potential showed a non-uniform distribution of fluorescent DNA, with small local areas of high

intensity which suggests a similar heterogeneity in DNA packing density for both

immobilisation procedures although much more homogeneous layers were obtained with the

second procedure. These measurements are based on the non-radiative energy transfer from the

excited dye molecule, attached to the DNA’s top end, to the metal. These brighter areas, named

as “hotspots”, corresponding to 10 to 30 % of the surface in the Tarlov immobilisation

procedure, were explained by the formation of aggregates with a significant amount located far

from the surface, meaning not covalently bound or non-specifically adsorbed. The central image

presented on Figure 1-15 corresponds to a fluorescence image of a gold surface modified by

MCH/ssDNA at open circuit potential and the associated sketches illustrate the presumed

origins of the heterogeneous fluorescent features observed.


22

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.

More recently, using the technique of in situ epifluorescence microscopy developed by the

group of Bizzotto, our lab investigated the heterogeneous nature of mixed SAMs of DNA and

mercaptobutanol [104]. Three types of procedures of immobilisation, namely DNA followed

by MCB adsorption, MCB followed by DNA adsorption and simultaneous adsorption of MCB

and DNA have been considered. Each of them led to some degree of heterogeneity.

Furthermore, it has been shown that aggregates remained on the electrode surface after a first

reductive desorption cycle, indicating the high stability of these features. In the absence of

diluent, an increase of the concentration of DNA in the solution of immobilisation leads to more

numerous and larger aggregates. The simultaneous recording of the capacitance and

fluorescence intensity as a function of the applied potential through reductive desorption


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].

Boncheva and Vogel [72], also considered the self-assembly of pure peptide layers which

were either constituted of amphipathic or hydrophobic helices. They were able to control the

orientation of the helices by choosing a particular adsorption procedure. While Langmuir-

Blodgett transferred monolayers were oriented parallel to the gold surface, self-assembled one

were oriented perpendicularly. Selecting the appropriate amino acid sequence, it is possible to

create tailor-made peptide layers of predefined conformation, α-helix or β-strand, orientation

and flexibility. Once a suitable sequence has been chosen, the molecular orientation of the

peptides at the interface may be controlled by mutual interactions between hydrophilic and

hydrophobic neighbouring peptides and the interfacial region, namely, the surface of a solid

support. The direction of the molecule at the surface can be controlled by the choice of a specific

binding group such as sulphur for anchoring on gold or silver. This statement was confirmed

by STM measurements performed by Davis et al. [75]. Using SPR and reflection-absorption

FTIR they were also able to study the molecular density of the peptide layers and their

molecular conformation and orientation on a solid support. They showed the influence of the

interface on the orientation of the polypeptide by comparing the behaviour of peptide

monolayers on water and after LB transfer to a hydrophilic gold surface. At the air/water

interface, both the hydrophobic and amphipathic helices were oriented parallel to the interface.

The absence of re-orientation of the hydrophobic peptide indicates that the conformation and


24

interfacial orientation of the peptides can be preserved upon LB transfer to the gold substrate.

Upon self-assembly, the hydrophobic peptide adopted a perpendicular orientation whereas the

amphipathic peptide kept its parallel orientation. The facts demonstrate the influence of the

choice of the peptide sequence for molecular orientation. It was also shown that the more

densely packed layers were obtained by self-assembly compared to LB transferred films.

Gatto et al. also studied self-assembled monolayers formed by helical peptides. The use of

Cα-tetrasubstituted amino acids allowed them to obtain rigid helical oligopeptides able to form

densely packed layers [109, 110]. Using helix-helix macrodipole interactions, they were able

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].

Considering the increasing attention paid to the formation of well-organised and

homogeneous layers, we decided to focus part of this work on the organisation of peptide

monolayers resulting from a series of immobilisation procedures using in situ fluorescence

microscopy.


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-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].

The ability of MDM2 to inactivate p53 relies on a direct physical interaction between the

two proteins. The regulation of the levels of MDM2 and p53 in the cell proceeds via a negative

feedback loop where p53 induces MDM2 expression whereas MDM2 represses p53 activity.

This cycle is considered as a central mechanism for modulating p53 activity in normal cells, in

absence of stress. Under normal conditions, low cellular levels of p53 are ensured through

MDM2 ubiquitination and associated degradation. In response to cellular stress, p53

degradation is reduced, and p53 is accumulated in the cell. Once its mission has been completed,

p53 transcriptionally activates the production of MDM2. The E3 ubiquitin ligase will then form

a tight complex with p53 to inhibit its transcriptional activity and promote p53 degradation via

multi-ubiquitination events on multiple p53 lysine residues. Once an appropriate level of p53

is reached in the cell, MDM2 proceeds to an auto-ubiquitination and is sent to the degradation

path. As a result, overexpression of MDM2 results in lower levels of p53, suppressing its

protective tumour activity. Binding of MDM2 to p53 is essential for this effect as the use of

inhibitors revealed that p53 is targeted for degradation by the proteasome. The analysis of

different human cancers in tumour samples indicates that MDM2 is overexpressed in 5 to 10 %


26

of human cancers [130-133]. Figure 1-16 presents the regulatory feedback cycle governing the

activity of p53 and MDM2.

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.

As it has been mentioned above, the ability of MDM2 to inactivate p53 relies on a direct

interaction between them. It has been shown that p53 binds to MDM2 via its transactivation

domain. Figure 1-17 presents the location of the structural features of p53 and human MDM2.


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].

Upon interaction, the MDM2 N-terminal domain forms a twisted trough, presenting a cleft

of about 25 Å long, 10 Å wide and 10 Å deep, lined with hydrophobic amino acids. The sides

of the cleft are formed by two helices whereas two shorter helices constitute the bottom. A pair

of three-stranded β sheets cover each end. Figure 1-19 illustrates the formation of the cleft.

When the p53 interaction sequence approaches MDM2, it forms an amphipathic α helix of about

2.5 turns, followed by 3 residues. The interaction of this α helix of p53 with the hydrophobic

cleft of MDM2 constitutes the primary contact.


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.

Figure 1-19: Structure of the p53 peptide (yellow) and MDM2 cleft (blue) complex [137].

.


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,

the charge transfer resistance gradually increases with the concentration of MDM2 indicating

that the binding of MDM2 to the antibody-modified electrode blocks the electron transfer

between the redox probe and the electrode surface. A detection limit of 0.29 pg mL-1 was

reached. Figure 1-20 illustrates their approach and obtained data.


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].

Li et al. also prepared an electrochemical sensor for the detection of MDM2 but with a

rather different approach [156]. They used a peptide, selected by combinatorial screening

(MUA-TSFAEYWNLLSP), able to bind to a low molecular weight probe. This molecule,

designed as an initiator of a signal amplification process, more precisely a photo-catalyst, binds

the peptide without interfering in the protein-peptide binding affinity. Their strategy is

illustrated at Figure 1-21.


31

Figure 1-21: Illustration of the principle of the electrochemical immunosensor for the protein

MDM2 developed by Li et al. [156].

Initially, the complex probe selectively binds the MDM2-free peptides immobilised on a gold

substrate via host-guest interaction with the aromatic side chain of the peptide. Under potential

control, the photo-catalytic probe is released in solution and, under UV excitation, catalyses the

cleavage of a dsDNA immobilised on a second gold electrode via the generation of hydroxyl

free radicals. The binding of the target protein can then be quantified via the remainder of

dsDNA immobilised on the second electrode after photo-cleavage using methylene blue as

redox marker. Indeed, as the amount of dsDNA initially immobilised is fixed, and as the

amount of probes bound with the MDM2-free peptides is inversely proportional to the amount

of MDM2, the amount of remaining dsDNA after cleavage is proportional to the amount of

MDM2. Figure 1-22 illustrates square wave voltammetry data obtained at the dsDNA modified

electrode for the measurement of MDM2 concentration.


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].

Another interesting aspect of gold is its ability to bind thiols with high affinity in order to form

self-assembled monolayers [54]. Furthermore, it does not undergo any unusual reactions with

thiols namely the formation of a substitutional sulfide interface as it can be observed on

palladium [64]. The high affinity of thiols for gold also allows the displacement of non-

specifically adsorbed species. Finally, since gold is one of the most studied substrate for the

formation of self-assembled monolayer, it will allows us to confront our results with literature.


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).

From the considerations above, a probe composed of the amino acid residues 12-26 has

been selected in this work. The truncation at residue 26 has been chosen from the higher binding

constant obtained by Schon et al. for shorter peptides. Residues 12 to 16 have been retained to

keep the residues 17-26 away from the electrode surface, enabling a better accessibility of the

target during the recognition process. Although this three amino acids elongation might reduce

the binding constant compared to the p53(15-29), we don’t expect it to suppress the affinity.


34

For immobilisation purpose, the sequence has been modified by a cysteine residue at the N-

terminal to allow the formation of a Au-S covalent bond with the substrate.

1.6.4 Working plan

In a first part of this thesis, we propose a label-free detection method of the protein MDM2

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.

The sensor performances directly depend on the monolayer properties. It is, therefore,

essential to obtain a detailed characterisation of the monolayer. For this purpose, we will, as a

second part of this thesis, focus on the formation and characterisation of the peptide-based

recognition layer alone or co-assembled with 4-mercaptobutan-1-ol, immobilised on gold

electrodes via self-assembly. A first, a standard electrochemical characterisation, in the

presence and absence of redox markers, of the various films formed has been performed,

allowing a preliminary study of the influence of the immobilisation procedure on the resulting

layer. A close collaboration with Professor Bizzotto from the University of British Columbia,

allowed us to implement, at the Université Libre de Bruxelles, a unique in situ epifluorescence

microscopy technique, he had elaborated. This implementation has been performed, thanks to

the access to an epifluorescence microscope located in the laboratory of Professor Michele

Sferrazza, of the Physics Department, at ULB. This technique, based on the simultaneous record

of electrochemical data and fluorescence images, in a specifically designed electrochemical

cell, will allow us to obtain a localised information of the organisation of our peptide layers.

This information constitute a very interesting complement to electrochemical data, which only

gives us an average information of what is happening on the surface. Atomic Force Microscopy

measurements performed in collaboration with Dr Philippe Leclère, from the Université de

Mons, will complete the characterisation of the organisation of the layers achieved through self-

assembly.

Chapter 2 Experimental

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.

The electrochemical cell, shown on Figure 2-1 is equipped with a Teflon lid with five openings

allowing the introduction of the electrodes, the nitrogen stream and the addition of a reagent

during the experiment if necessary.


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

reproducible cyclic voltammograms were recorded. Figure 2-2 presents a typical

characterisation curve of gold allowing to evaluate the quality of the substrate (black curve).


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.

The counter electrode, consisting in a large area platinum grid, was cleaned by heating in

a flame to red glow. The reference electrode was a Ag|AgCl|sat. KCl (XR 300 from Radiometer

or 6.0726.110 from Metrohm). In this work, all potentials are referred to this reference

electrode.


38

2.1.2 Cyclic voltammetry

Voltammetry is commonly employed for the study of electrode processes such as electron

transfer or adsorption phenomena. This electrochemical method, based on the measure of the

current resulting from the application of a potential at the working electrode, may provide

qualitative and quantitative information on the analyte.

More particularly, in the case of cyclic voltammetry, the applied potential varies linearly

between two chosen limits, E1 and E2. The potential program, called triangular in cyclic

voltammetry, is characterised by a scan rate υ = dE/dt. Figure 2-3 shows the potential profile

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.

𝐼 = 𝐼𝐹 + 𝐼𝐶 (2.1)

with,

𝐼𝐹 =𝑑𝑄𝑓

𝑑𝑡 (2.2)

and

𝐼𝐶 = 𝐶𝜐 (2.3)


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].

For a reversible process, the difference between the potential of the anodic and cathodic peaks

at 25° C is given by:

∆𝐸𝑝 = 𝐸𝑝𝐴 − 𝐸𝑝

𝐶 = 2.303 𝑅𝑇

𝑛𝐹=

59

𝑛 𝑚𝑉 (2.5)


40

where 𝐸𝑝𝐴and𝐸𝑝

𝐶 are respectively the anodic and cathodic peak potentials.

For an irreversible system at 25 °C:

𝐼𝑝 = 2.99 × 105𝑛3/2𝛼1/2𝐴𝐷1/2𝑐∞𝜐1/2 (2.6)

where α is the transfer coefficient (0<α<1), the other terms are the same as in equation 2.4.

In the case of a reversible process where the electroactive species are adsorbed at the electrode,

the peak current is given by:

𝐼𝑝 =

𝑛2𝐹2

4𝑅𝑇𝐴 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

2.718 𝑅𝑇𝐴 Γ 𝜐

(2.8)


41

According to the dependence of the peak current on the scan rate, it is possible to discriminate

whether an electron transfer process is controlled by the adsorption or diffusion of the

electroactive species. Indeed, one can note that, when the redox process is controlled by

diffusion, the intensity of the peak current evolves as a function of the square root of the scan

rate whereas a control by adsorption induces a linear dependence on the scan rate.


42

2.1.3 Chronoamperometry and chronocoulometry

Chronoamperometry consists in the application of a potential step from an initial value E1,

generally being in a region where no faradaic reaction occurs, to a final value E2 chosen in a

region where the process of interest takes place. The relaxation of the system following the

potential perturbation is recorded as a measure of the current I(t) versus time during a defined

time period.

The integration of the I(t) curve over time provides the charge Q(t) as a function of time. The

integrated analogue of chronoamperometry is chronocoulometry. In some cases, relevant to the

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.

The data presented in this work result from chronoamperometry measurements. The charge has

been obtained by subsequent integration.


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, �̇� and 𝐼,̇ rotating

at the same frequency as shown on Figure 2-7.

Figure 2-7 : Phasor diagram showing the relationship between alternating current and voltage

signals at frequency ω [162].


44

The two separate phasors, �̇� and 𝐼,̇ are generally not in phase and are separated by a phase angle

Φ. �̇� is usually taken as a reference signal and Φ is measured with respect to it. This latter is

constant since the relationship between two phasors remains constant as they rotate at the same

frequency. Therefore, phasors are usually plotted as vectors having a common origin and

separated by the appropriate angle.

The alternating voltage and current phasors are linked by the general equation:

�̇� = 𝐼�̇� (2.11)

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,…).

A frequently used circuit is the Randles equivalent circuit presented on Figure 2-8. It models

interfacial electrochemical reactions with a semi-infinite linear diffusion of electroactive

species at electrodes.


45

Figure 2-8: Randles equivalent circuit.

The resistance Rs is associated with the resistance of the solution, Cdl with the double layer

capacitance, Rct is the charge transfer resistance and ZW is the Warburg impedance. Depending

on the system, variations of the circuit can be observed such as the replacement of the double

layer capacitance by a constant phase element Qdl which will be defined in section 4.1.


46

2.2 Quartz Crystal Microbalance

Quartz crystal microbalance (QCM) is a sensitive device able to measure variations in mass

at the electrode surface based on small variations of the resonance frequency of a piezoelectric

crystal. The method is based on the converse piezoelectric effect corresponding to the

occurrence of a mechanical deformation upon application of an external electric field through

a crystal without center of symmetry, the resonator.

The quartz crystal is cut in such a way that a mechanical strain creates a permanent dipole

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 =

−2𝑓02 ∆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), µQ the shear modulus of the quartz (2.947 1011 g s-2 cm-1), ρQ the quartz

density (2.648 g cm-3) and S the Sauerbrey constant [164].


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)

Figure 2-9 : (a) Quartz crystal microbalance; (b) flow cell; (c) gold coated quartz crystal.


48

2.3 Atomic Force Microscopy

Atomic force microscopy (AFM) is a scanning probe technique providing surface

information on the nanoscale by measuring repulsive and attractive forces between a probe and

a surface. The principle is based on the short distance interactions between the sample and the

sharp probe supported on a flexible cantilever. The motion of the probe across the surface

sample is controlled by a piezoelectric crystal. A laser beam is reflected by the cantilever

towards a photodiode that detects the deflection of the probe as sketched in Figure 2-10.

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).

In this work, AFM measurements were operated in a non-contact mode with a silicon

nitride tip using a multimode microscope equipped with a Nanoscope V controller from Veeco

Instruments Inc.


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.

The electrochemical cell, arranged in a typical three-electrode setup, presents a 250 µm-

thick glass at its bottom optical window. It contains 7 entrances, one of them being connected

to a glass tube allowing nitrogen purge of the electrolyte and another to maintain the solution

under nitrogen during the experiments. A third port is used to insert the counter electrode which,

in this work, is a platinum wire. An integrated salt bridge allows the introduction of the


50

reference electrode, a KCl-saturated Ag|AgCl electrode, minimizing the contamination of the

electrolyte with the ions contained in the reference. Each potential given in this work refers to

this reference. The main port is centred and used to place the working electrode in the cell. The

remaining ports are closed with stoppers.

The working electrode is a polished polycrystalline gold bead prepared by melting the

extremity of a gold wire in a methane/oxygen flame. Prior to modification with the peptide,

each gold electrode was annealed in a methane/gas flame, cooled down for a few seconds in

air, and then quickly quenched in pure water. The beads were then electrochemically

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.

Two different setups including different microscopes, filter cubes, objectives and CCD

camera were used in this work, because part of the experiments (namely the characterisation of

the mixed layers of peptide and MCB prepared in one and two-step adsorption) were performed

in the lab of Professor Bizzotto at the University of British Columbia and the others

measurements at the Université libre de Bruxelles. Table 2-1 summarises the equipment used

respectively at UBC and ULB.


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.

Therefore, the bigger the numerical aperture, the best the resolution. The selected objectives

present quite small numerical aperture. However, our experiments require objectives

characterised by long working distances (WD). This parameter, defined as the distance from

the front lens element of the objective to the closest surface of the sample for which this latter

is in focus, has to be increased considering the electrochemical setup. Therefore, a compromise

has to be made between the two parameters, R and WD. Some objectives as the 50 × CFI LU

Plan EPI ELWD are specially designed to present a high numerical aperture compared to the

working distance. The acronym ELWD stands for “extra long working distance”. This type of

objective is often used on reflected light and inverted microscopes.


52

Calibrations of the size of the fluorescence images have been performed using FocalCheckTM

fluorescence test slides. Table 2-2 presents the calibration of the CCD Cameras.

Table 2-2 : Calibration of the CCD Cameras.

EvolveTM 512 EMCCD Camera Clara Interline CCD Camera

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.

In both setups, a succession of fixed potentials was applied every 25 mV as shown on Figure

2-12.


53

Figure 2-12 : Illustration of the potential programming and associate triggering used during in

situ fluorescence microscopy measurements


54

2.5 Chemicals

The water, used as solvent is Milli-Q water (Millipore). It is free from organic traces and

presents a resistivity > 18.2 MΩ cm.

The nitrogen (purity > 99.9 %), supplied from Air Liquide, used to remove oxygen from the

solutions, contains less than 0.0005 % oxygen.

The salts used to prepare the Tris buffer are purchased from Alfa Aesar:

Tris(hydroxymethyl)aminomethane hydrochloride, 99+% Tris(hydroxymethyl)amino-

methane, ACS, 99.8-100%

The salt used to prepare the phosphate buffer; Na2HPO4.2H2O and NaH2PO4.H2O, are of pro

analysi grade and supplied from Merck.

The perchloric acid 70 % is purchased from Merck and is of Suprapur quality.

The potassium hexacyanoferrate (III) and potassium hexacyanoferrate (II) trihydrate are of

European Pharmacopoeia Reagent grade and supplied from Merck.

The hexaammineruthenium(III) chloride is an Alfa Aesar product from Johnson Matthey

GmbH company (Ru 32.1 % min).

The 4-mercaptobutan-1-ol (MCB) purchased from Fulka presented a 97 % purity and was

stored at 4 °C.

The peptide aptamer sequences have been synthesised and purified by Eurogentec S.A. Europe.

Purifications have been performed by high performance liquid chromatography. The selected

peptide aptamers are based on the amino acids sequence of the protein p53 involved in the

interaction with the protein MDM2. For anchoring on a gold substrate the sequence has been

modified with a cysteine residue and for fluorescence measurements, the sequence has been

elongated with three amino acids and labelled with a 5-carboxyfluorescein tag. Table 2-3

summarises the peptide aptamer sequences used in this work.

Table 2-3 : Selected peptide aptamer sequences of the protein p53.

Peptide aptamer sequences

Ap-cys-p53 H2N-CPPLSQETFSDLWKLL-COOH

Ap-cys-p53-fluo H2N-CPPLSQETFSDLWKLLPAK(5-FAM)-CONH2

Ap-p53-fluo H2N-PPLSQETFSDLWKLLPAK(5-FAM)-CONH2


55

The MDM2 human recombinant target protein (expressed in E. coli) has been purchased from

Sigma-Aldrich and supplied as a liquid solution whose solvent was composed of 20 mM Tris-

HCl pH 8, 20 % glycerol, 100 mM KCl, 1 mM DTT, 0.2 mM EDTA. To prevent interferences

of these additionnal compounds, particularly DTT during the experiments, the aliquot was

further purified on a PD MidiTrap G-10 column (GE Healthcare). After equilibration of the

column with 20 mM Tris-HCl pH 8, 1 mL of the sample has been added until it completely

entered the packed bed followed by 0.7 mL of the buffer. After discarding the flow-through,

1.2 mL of buffer were used for elution and collection of the protein sample in a tube. The

resulting concentration of protein was determined using a Pierce BCA Protein Assay procedure

based on a Biuret reaction. The sample was stored at -80 °C.

Albumin from bovine serum ≥ 99 % purified by agarose gel electrophoresis was purchased

from Sigma-Aldrich and stored at 4 °C.

Cytochrome c from bovine heart ≥ 95 % was purchased from Sigma-Aldrich and stored

at -20° C.

Fibrinogen from human plasma ≥ 90 % was purchased from Calbiochem and stored at 4 °C.

Chapter 3 Electrochemical characterisation of the

immobilisation of the peptide aptamer probe on gold

57

Chapter 3. Electrochemical characterisation of the


3.1 Formation of the self-assembled monolayers

The immobilisation of the probe on the electrode surface is one of the key aspects in the

elaboration of a biorecognition interface. Many features that have to be taken into account have

been widely underlined in the literature such as the amount of immobilised probe for a highly

sensitive detection signal for the analytical process as well as the appropriate density of probe

to allow a sufficient accessibility of the immobilised molecules for the target. In this context,

attention has been drawn to the formation of homogenous layers [80, 99, 103, 104, 109, 112,

75, 165]. Furthermore, the modified interface has to present a good selectivity for the target

molecule.

The immobilisation process consisting in the anchoring of one of the extremity of the peptide

probe to the surface is commonly used and considered as a convenient method. This procedure

allows some control of the density of the probe and preserve a conformational freedom of the

probe for the recognition event.

In this work, we propose a self-assembled immobilisation procedure of a peptide aptamer. This

probe is based on the interaction sequence of the p53 tumour suppressor protein with the

oncoprotein MDM2 and more particularly the amino acids 12 to 26. The peptide sequence has

been modified by a cysteine residue to allow the chemisorption of the probe on the gold

substrate through the formation of a Au-S bond. In the following work, we will refer to the

probe sequence as “Ap-cys-p53”.

The formation of the biorecognition layer has been realised by self-assembly of the thiolated

peptide probe on a polycrystalline gold electrode.

Two ways of immobilisation have been considered in this work,

- a one-step coadsorption procedure in which a hydroxyalkanethiol, the 4-

mercaptobutan-1-ol (MCB) and the p53 based thiolated peptide aptamer are

simultaneously adsorbed on the gold surface as can be seen on Figure 3-1 (a).



58

- a two-step adsorption procedure in which the p53 peptide probe is firstly

immobilised followed by the “passivation” of the gold surface with 4-

mercaptobutan-1-ol as can be seen on Figure 3-1 (b).

The following immobilisation conditions have been employed:

- a total concentration of thiolated probe and hydroxyalkanethiol maintained at

20 µM.

- a 50 µL immobilisation medium consisting in 1 M sodium phosphate buffer pH 7.4

- a long adsorption time (> 16 hours)

- different peptide/diluent molar ratios

The coadsorption of the biological probe with a hydroxyalkanethiol is commonly used in

the elaboration of a biorecognition interface. It prevents the non-specific adsorption of

biological molecules on the gold surface. It also allows the probe to straighten up to be

accessible to the target. Figure 3-1 illustrates both ways of adsorption. The self-assembly occurs

through simple immersion of the gold substrate in a buffered solution containing a known

concentration of the thiolated species.

(a) One-step coadsorption

(b) Two-step consecutive adsorption

Figure 3-1 : Illustration of the immobilisation procedures through (a) one-step coadsorption

and (b) two-step consecutive adsorption.



59

3.2 Evidence of the immobilisation of the thiolated

molecules

The presence of immobilised molecules at the electrode surface can be detected by

evidencing the reductive desorption of these species. Figure 3-2 shows the evolution of the

cyclic voltammograms of a Ap-cys-p53/MCB (2/3) modified gold electrode in one-step

coadsorption while cycling the potential between -0.8 V and +0.1 V. A modification of the

shape of the voltammograms, associated with the reductive desorption of the thiolated species

immobilised on the electrode surface, can be observed over cycling. A continuous decrease in

the intensity of both the cathodic and anodic responses is noticed at each cycle indicating that

a smaller amount of thiolated molecules is adsorbed on the surface. This decrease originates in

the diffusion from the electrode surface to the bulk of the desorbed thiolated peptide and MCB

at potentials more negative than -0.5 V. The molecules keep diffusing as long as the potential

has not reached a value positive enough to allow the re-adsorption of the thiolated species and

only those being close enough to the electrode will be re-adsorbed.

-0.8 -0.6 -0.4 -0.2 0.0 0.2

-15

-10

-5

0

5

j /

µA

.cm

-2

E vs Ag|AgCl / V

scan 1

scan 2

scan 4

scan 7

scan 10

Start potential

Figure 3-2: Evolution of the cyclic voltammograms of a coadsorbed Ap-cys-p53/MCB (2/3)

modified electrode recorded in 10 mM Tris-HCl – pH 7.4; υ = 50 mV s-1.

Figure 3-3 presents the cyclic voltammograms recorded in the ideally polarisable domain,

before and after an overnight immersion in a 1 M phosphate buffer solution pH 7.4 containing

a 20 µM mixture of Ap-cys-p53/MCB in proportion 2/3 (coadsorption procedure). Whereas no

faradaic current is observed on the curve corresponding to the bare gold, the presence of the



60

layer after the immobilisation is evidenced by the appearance of a cathodic peak at -0.7 V

corresponding to the reductive desorption of the thiolated species immobilised on the electrode

surface. The potential sweep is inverted when the peak corresponding to the reduction of the

electrolyte appears. On the backward sweep, a smaller peak, originating from the partial re-

adsorption of the thiolated species, is observed around -0.53 V.

-0.8 -0.6 -0.4 -0.2 0.0 0.2

-20

-15

-10

-5

0

5

-0.8 -0.6 -0.4 -0.2 0.0 0.2

-15

-10

-5

0

5 (b)

j /

µA

.cm

-2

E vs Ag|AgCl / V

(a)j

/ µ

A.c

m-2

E vs Ag|AgCl / V

Start potential

Figure 3-3 : Cyclic voltammograms of a polycrystalline gold electrode before (a) and after (b)

immersion in a 20 µM solution of Ap-cys-p53/MCB (2/3) recorded in 10 mM Tris-HCl -

pH 7.4, υ = 50 mV s-1.

At pH 7.4, desorption occurs according to the following reaction:

𝑅𝑆 − 𝐴𝑢 + 1𝑒− ⇄ 𝑅𝑆− + 𝐴𝑢



61

The mechanism of reductive desorption involving the transfer of one electron per adsorbed

molecule, the total amount of adsorbed self-assembled thiolated molecules can be determined,

after the first cycle, through the integration of the cathodic peak to obtain the faradaic charge

according to equation 3.1:

𝜎𝑓 = 𝑛𝐹Γ𝑡𝑜𝑡 (3.1)

With σf the charge density, n the number of electron transferred (n assumed as being equal to

1 in the present case), F the Faraday constant, Γtot the total surface concentration in thiolated

molecules, ΓMCB + ΓAp-cys-p53.

The integration of the cathodic peak of the cyclic voltammogram of the coadsorbed Ap-

cys-p53/MCB 2/3 modified electrode presented in Figure 3-3 leads, after subtraction of the

capacitive current, to a faradaic charge of 45.5 ± 0.9 µC cm-2 so that the total surface

concentration in thiols amounts to 4.7±0.1 10-10 mol cm-2.

In the case of the two-step adsorption procedure in which the peptide probe is adsorbed on the

electrode surface overnight prior to the MCB (1 hour), the same shape and behaviour is

observed for the cyclic voltammograms associated with the reductive desorption. However, the

layer presents a lower stability since it already desorbs at -0.6 V compared to -0.7 V for the

one-step adsorption procedure. For a two-step adsorption, with a same peptide/MCB ratio (Ap-

cys-p53/MCB 2/3), a faradaic reduction charge of 41.2 ± 0.7 µC cm-2 is measured giving

therefore a total surface concentration of 4.2±0.1 10-10 mol cm-2. These data indicates that both

ways of immobilisation lead to a comparable amount of adsorbed thiolated molecules. These

results can be compared with those obtained for compact self-assembled alkanethiols

monolayers where concentrations of 7.6 10-10 mol cm-2 are obtained on Au(111) [69, 166]. The

smaller value obtained in this work is related to the bigger size of the peptide molecule

compared to an alkanethiol.

Although these reductive desorption measurements evidence the immobilisation of a layer of

thiols, it only gives a total thiol concentration and does not unarguably prove the adsorption of

the peptide on the electrode surface, even though the smaller total superficial excess compared

to a full layer of alkanethiol supports this hypothesis. Therefore, we need additional approaches

to provide further evidence of the biomolecule adsorption, as well as to quantify its amount.



62

3.3 Interaction between the [Ru(NH3)6]3+ complex and

the peptide probe

3.3.1. Evidence of the interaction between the complex and the

peptide probe

The characterisation of the monolayer requires the control and determination of the amount

of probe immobilised on the surface. In the field of biosensing, this quantification is often

processed through the use of radioactive or fluorescent marker [167, 168, 169, 170].

Nevertheless, Steel et al. developed an easy-to-use electrochemical quantification to monitor

the amount of DNA oligonucleotides adsorbed on a gold surface [77]. The surface density of

DNA was determined by taking advantage of the electrostatic interaction of the positively

charged hexaammineruthenium(III) redox complex with the negatively charged phosphate

backbone of DNA. In this technique, a DNA modified electrode was soaked in a low ionic

strength electrolyte containing the cationic redox marker which can exchange for the native

counterions associated with the negative phosphate residues of the oligonucleotides. From

chronocoulometric measurements one can calculate the amount of redox marker in electrostatic

interaction with DNA. At saturation coverage of the redox marker, assuming a complete

compensation of the DNA residues by redox cations, the surface density of the probe can be

calculated. This method has also been developed by Yu et al. who were able to evaluate the

surface densities of both single and double-stranded molecules via the integration of the peak

for reduction of [Ru(NH3)6]3+ bound electrostatically to DNA as well as the binding constant

and electron transfer rate constant of [Ru(NH3)6]3+ on DNA modified electrodes [171, 172]. In

an extended view, Steichen et al. showed that the method, developed by Tarlov, can be applied

to anionic self-assembled monolayers namely on compact monolayers of 2-

mercaptobenzimidazole-5-sulfonate (MBIS) on gold [173].

As, in this work, we will be working with oligopeptides instead of oligonucleotides, we

will remain cautious about the results obtained from the quantification based on the

hexaammineruthenium(III). Although this method can give us an information, firstly, on the

presence of the probe on the surface and, secondly, on the relative amount of immobilised

peptide regarding the adsorption procedure, the presence of both negative and positive charges

on the peptide can have an influence on the results since intramolecular electrostatic interactions

may occur. However, at the working pH of this work, 7.4, our peptide probe presents a positive

charge on the lateral chain of the lysine residue and a negative charge on the lateral chains of

the aspartic acid residue and glutamic acid residue. Besides lateral chains, the peptide also

presents both a negative and a positive charge on the C- and N-terminal position respectively,

resulting therefore in a globally negatively charged probe allowing the use of [Ru(NH3)6]3+ as

dissolved marker.



63

The interaction of the hexaammineruthenium(III) with the immobilised peptide probe is

evidenced by the cyclic voltammogram of a gold electrode exclusively modified with the

peptide presented on Figure 3-4.

-0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3-15

-10

-5

0

5

10j

/ µ

A.c

m-2

E vs Ag|AgCl / V

(B)

(A)

Figure 3-4 : Cyclic voltammogram of a 20 µM Ap-cys-p53 modified gold electrode in 10 mM

Tris-HCl – pH 7.4 -50 µM [Ru(NH3)6]3+; υ = 50 mV s-1.

Two cathodic responses are observed, a first peak (A) at -0.15 V corresponding to the diffusion

of the electroactive species to the electrode is followed by a post-peak (B) at more negative

potentials, around -0.3 V. This voltammogram characteristic shape witnesses the adsorption of

dissolved electroactive species at the electrochemical interface [174]. The post-peak results

from the greater stability with respect to the reduction of the adsorbed electroactive species as

compared to the dissolved ones. These data confirm the adsorption of the [Ru(NH3)6]3+ at the

modified electrode, and this can be assigned to the successful immobilisation of the peptide

probe.

Cyclic voltammograms recorded at different scan rates support the adsorption of

[Ru(NH3)6]3+ at the modified electrode. By plotting the current associated with both peaks as a

function of the scan rate (Figure 3-5), it is found that the intensity of the peak A is proportional

to the square root of the scan rate, indicating a diffusion controlled process. However, a linear

relation of the intensity as a function of the scan rate is observed for peak B, confirming the

reduction of the adsorbed electroactive species.



64

0 50 100 150 2000

2

4

6

8

10

12

14

16 peak A

peak B

j p /

µA

.cm

-2

/ mV.s-1

Figure 3-5: Peak current densities of the peaks A and B as a function of the scan rate for the

[Ru(NH3)6]3+ on a Ap-cys-p53/MCB modified electrode.

Figure 3-6 compares the cyclic voltammograms recorded for mixed layers of Ap-cys-p53/MCB

immobilised by two-step adsorption (curve a) and coadsorption (curve b) procedures.

Figure 3-6: Cyclic voltammograms of a mixed layer Ap-cys-p53/MCB modified gold

electrode by two-step adsorption (a) and coadsorption (b) in 10 mM Tris-HCl – pH 7.4 -50

µM [Ru(NH3)6]3+ ; υ = 150 mV s-1.

It can be noticed that the post-peak associated with the [Ru(NH3)6]3+ adsorbed on the peptide is

markedly smaller on curve (b), corresponding to the coadsorption of the peptide with MCB,

-0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3-15

-10

-5

0

5

10

-0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1-15

-10

-5

0

5

10(a)

j/µ

A.c

m-2

E vs Ag|AgCl /V

j/µ

A.c

m-2

E vs Ag|AgCl/V

(b)



65

than on curve (a). This variation could indicate that coadsorption leads to monolayers less

concentrated in peptide compared to a two-step adsorption.

In principle, it should be possible to quantify the amount of adsorbed [Ru(NH3)6]3+ through

the integration of the post-peak, provided it is sufficiently separated from the main wave.

Indeed, as it has been mentioned earlier, Yu et al. based the quantification of the surface density

in DNA on the integration of the peak for reduction of [Ru(NH3)6]3+ electrostatically bound to

DNA [171]. In practice, it is usually complicated to subtract the baseline and to correct for

double layer charging. Therefore, we will use chronocoulometry which allow the determination

of the amount of adsorbed reactant independently of the relative position of the dissolved and

adsorbed marker reduction as well as the kinetics of the reaction.

From chronocoulometric data, we can obtain the charge density σ, measured as a function of

time (t > 0) as it is expressed in equation 3.2 [174]:

𝜎 =

2𝜋𝐷𝑂1/2

𝐶𝑂

𝜋1/2𝑡1/2 + 𝜎𝑑𝑙 + 𝑛𝐹𝛤𝑂 (3.2)

with n the number of electrons involved in the reduction of the redox species, DO its diffusion

coefficient, CO its bulk concentration, σdl the charge associated to the double layer, ΓO the

surface concentration of redox cations adsorbed at the electrochemical interface and F the

Faraday constant.

The first term of this equation, commonly referred to as the Cottrell contribution, represents the

charge associated with the redox process involving electroactive species diffusing to the

electrode. The second term is associated with the charge contribution due to the presence of an

electrical double layer at the electrode|solution interface. The third term, the one of interest in

this work, allows the quantification of the redox species adsorbed at the electrode.

Practically, the surface concentration of adsorbed redox cations is obtained in two stages in a

low ionic force buffer. A potential step programmation is applied from E1 to E2, firstly in the

absence of the redox complex to determine the charge of the double layer σdl and, secondly in

the presence of [Ru(NH3)6]3+. The same programmation is used in the present work.

The first potential E1 is chosen in a region where no faradaic reaction occurs, in the present case

+0.2 V. The choice of the second potential E2 is more complicated since it must be negative

enough so that all the adsorbed [Ru(NH3)6]3+ can be quantified but not too negative to avoid

the desorption of the monolayer. In this work, the second potential fulfilling both criteria

is -0.4 V.

Typical chronocoulometric curves of a Ap-cys-p53/MCB modified electrode in Tris-HCl 0.01

M-pH 7.4 in the absence and presence of 50 µM [Ru(NH3)6]3+ for a potential jump between

+0.2 V and -0.4 V are presented on Figure 3-7.



66

The surface concentration of adsorbed redox cations ΓO is obtained through the difference

between the extrapolation lines at t=0 of the charges measured in the presence and absence of

the [Ru(NH3)6]3+ complex.

0.0 0.2 0.4 0.6 0.8 1.00

2

4

6

8

10

12

14

16

18

20

22

| |

/ µ

C.c

m-2

t1/2

/ s1/2

without [Ru(NH3)

6]

3+

with [Ru(NH3)

6]

3+

Figure 3-7: Chronocoulometric curve of a coadsorbed Ap-cys-p53/MCB (2/3) in absence ( )

and presence ( ) of 50 µM [Ru(NH3)6]3+ in 10 mM Tris-HCl–pH 7.4.

E1= +0.2 V; E2=-0.4 V.

As we evidenced that the behaviour of the [Ru(NH3)6]3+ follows a process of adsorption relying

on an equilibrium at the interface between the modified electrode and the solution, we can

expect an influence of the concentration. Therefore, by varying the concentration in redox

complex in solution, we should be able to determine an adsorption isotherm of the [Ru(NH3)6]3+

in electrostatic interaction with the peptide and extract an adsorption constant. This

characterisation of the interaction will help us to support the use of the [Ru(NH3)6]3+ complex

as an analytical tool to evaluate the influence of different adsorption procedures on the probe

density in the self-assembled monolayers. Figure 3-8 presents the chronocoulometric data of a

two-step adsorption Ap-cys-p53/MCB (2/3) modified electrode in a 10 mM Tris-HCl pH 7.4

buffer solution containing different concentrations of [Ru(NH3)6]3+ varying from 0 to 100 µM.



67

0.0 0.2 0.4 0.6 0.8 1.00

5

10

15

20

25

30

||

/ µ

C.c

m-2

t1/2

/ s1/2

0 µM

1 µM

2 µM

5 µM

10 µM

15 µM

20 µM

25 µM

30 µM

40 µM

50 µM

60 µM

Figure 3-8: Chronocoulometric curves of a two-step adsorption Ap-cys-p53/MCB (2/3)

modified electrode recorded in 10 mM Tris-HCl-pH 7.4 containing varying concentrations in

[Ru(NH3)6]3+ from 0 µM to 60 µM. E1= +0.2 V ; E2=-0.4 V.

At lower concentrations, the extrapolation lines at t=0 show an increase of the amount of

adsorbed complex up to a certain concentration cO at which a maximum value is reached. A

further increase in concentration only leads to an increase of the slope of the line corresponding

to the Cottrell contribution. A curve showing the shape of an isotherm of adsorption is observed

when plotting the charge density of the adsorbed [Ru(NH3)6]3+ as a function of its concentration

(Figure 3-9).



68

0 10 20 30 40 50 60

0

2

4

6

8

10

12

|a

ds| /

µC

.cm

-2

[RuHex]/µM

Figure 3-9: Cathodic density of charge of the adsorbed [Ru(NH3)6]3+ as a function of the

concentration in [Ru(NH3)6]3+ in solution for a two-step adsorption Ap-cys-p53/MCB (2/3)

modified electrode in 10 mM Tris-HCl – pH 7.4. E1= +0.2 V; E2=-0.4 V.

The data are conveniently described by a Langmuir adsorption isotherm responding to

equation 3.3:

𝜎𝑎𝑑𝑠

𝜎𝑠𝑎𝑡 − 𝜎𝑎𝑑𝑠= 𝐾𝑎𝑑𝑠[𝑅𝑢𝐻𝑒𝑥] (3.3)

With [RuHex] the concentration of the species in solution, σads the charge involving the

adsorbed species, σsat the charge at saturation and Kads the adsorption constant [175].

This model assumes the equivalence of all sites of adsorption and the ability of each

molecule to bind independently from the occupation of the neighbouring sites. The validation

of the Langmuir adsorption isotherm is often assessed via its linearization and the equation 3.3

becomes,

[𝑅𝑢𝐻𝑒𝑥]

𝜎𝑎𝑑𝑠=

[𝑅𝑢𝐻𝑒𝑥]

𝜎𝑠𝑎𝑡+

1

𝐾𝑎𝑑𝑠𝜎𝑠𝑎𝑡 (3.4)

A linear graph of [𝑅𝑢𝐻𝑒𝑥]

𝜎𝑎𝑑𝑠 as a function of the concentration in [Ru(NH3)6]

3+ is in agreement with

the Langmuir model. The determination of Kads and σsat can then be graphically determined

respectively via the extrapolation of the line at x=0 and its slope. Figure 3-10 shows a linear

relation between the experimentally determined [𝑅𝑢𝐻𝑒𝑥]

𝜎𝑎𝑑𝑠 and the concentration in [Ru(NH3)6]

3+

in solution.



69

0 10 20 30 40 50 600

1

2

3

4

5

6

[Ru

Hex

]/

ad

s / µ

M.µ

C-1

.cm

2

[RuHex] / µM

Figure 3-10: Cathodic density of charge of the adsorbed [Ru(NH3)6]3+ as a function of the

concentration in [Ru(NH3)6]3+ in solution for a two-step adsorption Ap-cys-p53/MCB (2/3)

modified electrode in 10 mM Tris-HCl – pH 7.4 presented according to the linear form of the

Langmuir adsorption isotherm.

According to the conditions used in this work, 10 mM Tris-HCl–pH 7.4 buffer, the value of

adsorption constant obtained is 1.3 (± 0.6) 105 mol-1 L (n=3). This value reflect a strong

interaction between the complex and the peptide. Therefore, an analytical method based on this

interaction can be conceived.

3.3.2. Influence of the immobilisation procedure on the

[Ru(NH3)6]3+ concentration adsorbed at the

electrochemical interface

In the case of DNA, Tarlov et al. have shown that the quantification of oligonucleotides

immobilised in the SAM can be evaluated [77]. This quantification is based on a hypothesis

assuming that for diluted mixed monolayers all phosphate groups are associated with cationic

redox species so that the direct determination of DNA superficial excess is possible since a

complete compensation of charge corresponds to a 3 to 1 ratio (3 phosphate groups/ 1

[Ru(NH3)6]3+).

This hypothesis is more questionable in the present work since the peptide probe presents both

positive and negative charges allowing the presence of electrostatic interaction within the

peptide itself. Therefore, we propose here only to consider ΓO, the amount in redox cations

adsorbed at the electrochemical interface, rather than the peptide surface concentration.



70

Nevertheless, we will use the Tarlov method for a comparison purpose and consider the values

obtained for ΓO to compare how different procedures of adsorption will influence the density

of the probe assuming a similar accessibility of the probe irrespective of the immobilisation

conditions. Figure 3-11 presents the values of ΓO obtained for different mixed monolayers

prepared in varying the proportions of peptide and MCB in the immobilisation solution both in

a one-step coadsorption and a two-step adsorption procedure. For a constant concentration of

20 µM in thiolated molecules in the immobilisation solution, the mole fraction of peptide probe

has been varied and the amount of adsorbed [Ru(NH3)6]3+ has been measured by

chronocoulometry.

0.0 0.2 0.4 0.6 0.80

2

4

6

8

10

Coadsorption

Two-step adsorption

O /

10

-11m

ol.

cm-2

xpeptide

Figure 3-11: Surface concentration in [Ru(NH3)6]3+ , (n=3), in a mixed self-assembled

monolayer of Ap-cys-p53/MCB obtained by coadsorption (■) and two-step adsorption (●) as

a function of the mole fraction of Ap-cys-p53 present in the solution of immobilisation

recorded in 10 mM Tris-HCl-pH 7.4 in the presence of 50 µM [Ru(NH3)6]3+. E1= +0.2 V;

E2=-0.4 V.

We can notice on Figure 3-11 that the amount of adsorbed [Ru(NH3)6]3+ is constant for a

given immobilisation procedure irrespective of the amount of peptide probe in the solution.

Therefore, we might suggest that, for the concentration in thiols considered in this work, a

limiting coverage in Ap-cys-p53 is reached in the mixed monolayers. The mean value of the

surface concentration reaches 4.0 ± 0.2 10-11 mol cm-2 for the one-step coadsorption and

6.4 ± 0.2 10-11 mol cm-2 for the two-step adsorption. Comparing these values to those obtained

for the total surface concentration in thiols, respectively 4.7 ± 0.1 10-10 mol cm-² and

4.2 ± 0.1 1010 mol cm-², and considering a one to one ratio between the[Ru(NH3)6]3+ and the



71

peptide, we can estimate that about 10 % of the surface is covered by the peptide probe in one-

step coadsorption against 15 % for the two-step adsorption.

The amount of adsorbed [Ru(NH3)6]3+ however differs significantly between the two

procedures. Indeed, the [Ru(NH3)6]3+ surface concentration obtained for a mixed layer prepared

by coadsorption is 30 % lower than that obtained for a two-step adsorption. This trend has

already been evidenced in our lab for the immobilisation of mixed layers of oligonucleotides

and alkanethiols [104]. Some explanations can be found in the kinetics and thermodynamics of

adsorption of those species.

It is known that for thiols on gold, an initial adsorption step results in a surface coverage of

about 80 to 90 % at short times. This step is followed by a second stage at a much slower rate

during which the organisation of the SAM occurs. It is also known that for longer chains of

alkanethiols, the rate of the initial adsorption decreases, a phenomenon that is explained by the

slower diffusion of these thiols [70]. These informations may help us to interpret the difference

in peptide concentration for the two adsorption procedures.

In the case of the two-step adsorption, the peptide molecules are firstly self-assembled overnight

on the gold electrode surface through the thiol function of the cysteine residue. Over this period,

a maximum coverage of the peptide is reached. In addition to the covalently bonded molecules,

non specific adsorption of the amino acids on the substrate can also occurs. After the first

immobilisation step, MCB is put in contact with this initial layer. The peptide molecules

composing the SAM, when exposed to solutions containing other thiols, in this case MCB, may

exchange progressively. As the peptide is globally negatively charged, repulsions are expected

between molecules and the replacement of some of the peptide molecules by alkanethiols is

facilitated. Arinaga et al. evidenced this replacement of biomolecules, more specifically

oligonucleotides, by alkanethiols in real time by optical means [176]. They showed that the

mercaptohexanol induces the desorption of weakly adsorbed DNA and the reorientation of the

covalently attached strands.

During the one-step coadsorption, as both the peptide and the MCB are thiolated, a competition

directly occurs between these two species. Besides the charge difference, there is also a

difference in size between the two molecules. It is known that the adsorption process followed

a diffusion controlled Langmuir kinetics. The smaller MCB molecules, diffusing faster to the

electrode than the peptide, will rapidly cover a significant surface. It has been showed by Bain

et al. [55], that for long chain alkanethiols, the film thickness reached 80 to 90 % of its final

value after two minutes. It has also been showed by Subramanian et al. [71], that for

concentration in alkanethiols lower that 5 µM, 80 % coverage is reached after 500 s. The results

obtained here are consistent with the fact that, due to fast adsorption kinetics of the MCB on

the gold electrode, only a limited number of sites remain free for the thiolated peptide which

explains the smaller density of probe in the coadsorbed mixed monolayer compared to the one

obtained by the two-step adsorption.



72

3.4 Electrochemical behaviour of the redox couple

[Fe(CN)6]3-/4- in presence of the monolayers

As biomolecules are immobilised at the electrochemical interface, modifications in the

electrical double layer capacitance and in electron transfer kinetics are expected. The peptide

probe being globally negatively charged, its immobilisation generates an increase of the

negative charges at the electrochemical interface. It has been shown that in the presence of the

positively charged redox marker, [Ru(NH3)6]3+, electrostatic attraction occurred between the

peptide and the cation as evidenced by the appearance of an adsorption post-peak on the cyclic

voltammogramm presented on Figure 3-4. Therefore, using anionic redox markers in solution,

we expect to evidence electrostatic repulsions between the peptide monolayer and these

complexes. The redox couple [Fe(CN)6]3-/4- is frequently used to characterise the properties of

the self-assembled monolayers towards electron transfer. Figure 3-12 shows the cyclic

voltammograms of a polycrystalline gold electrode in the presence of [Fe(CN)6]3-/4- before and

after the immobilisation of a mixed layer Ap-cys-p53/MCB by coadsorption and two-step

adsorption. It also shows the electrochemical behaviour of the [Fe(CN)6]3-/4- towards a

monolayer exclusively composed of the peptide probe.

-0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6

-400

-300

-200

-100

0

100

200

300

400

j /

µA

.cm

-2

E vs Ag|Agcl / V

Bare gold

Coadsorbed peptide/MCB (2/3)

Two-step adsorption peptide/MCB (2/3)

Ap-cys-p53(12-26)

Figure 3-12 : Cyclic voltammograms recorded in a 50 mM phosphate buffer pH 7.4 in the

presence of 5 mM [Fe(CN)6]3-/4- 1/1, before (---) and after modification of a gold electrode by

coadsorption of 20 µM Ap-cys-p53/MCB (2/3) (---), two-step adsorption of 20 µM Ap-cys-

p53/MCB (2/3) (---) and adsorption of 20 µM of Ap-cys-p53 ( ) overnight ; υ = 50 mV s-1.



73

It can be seen that the [Fe(CN)6]3-/4- redox couple presents a nicely reversible behaviour on bare

gold. Upon immobilisation of the layers, the reversibility of the redox couple [Fe(CN)6]3-/4- is

affected. While the faradaic response remains highly reversible in the case of a coadsorbed

layer, a significant inhibition of the signal is observed for the films formed through a two-step

adsorption. Such a modification of electron transfer kinetics of redox molecule within films has

already been evidenced by Ceres et al. for DNA modified electrodes passivated by

mercaptohexanol. They showed that the permselectivity is related to the initial deposition

conditions, interduplex distances being dictated by the screening of the negative charges of the

phosphate backbone [177].

It is however very impressive to notice on Figure 3-12 that the redox peaks totally disappear

for a layer exclusively composed of the peptide probe.

It has been shown by Steichen et al. that self-assembly of mercaptobutanol on gold has no

significant influence on the redox process of [Fe(CN)6]3-/4- [79]. Therefore, the inhibition

observed here is clearly associated with the presence of the negatively charged peptide aptamer.

We have seen earlier that coadsorption was leading to a lower density of the aptamer in the

mixed film allowing thus the mercaptobutanol to preserve channels through the film where the

redox couple kinetics is merely affected. The higher density of peptide aptamer in the two-step

adsorption procedure increases the amount of negative charges at the electrode surface and

further hinders the electron transfer. This effect is reinforced for the layer exclusively composed

of peptide, since the absence of mercaptobutanol removes any facile access to the electrode.

The negatively charged peptide layer forms thus a barrier to the electronic transfer, and totally

inhibits the signal.

The insulating property of peptide films has already been evidenced for helicoidal peptides

by Gatto et al. who used Cα-tetrasubstituted amino acids behaving as rigid helical oligopeptides

able to form densely packed layers [109, 110]. Although the transactivation domain of the p53

is supposed to be intrinsically disordered and to form a helicoidal structure only under

interaction with the MDM2 target protein, a multidimensional NMR study carried by Lee et al.,

has shown that this domain is populated by an amphipathic helix and two nascent turns. The

helix is formed by residues Thr18–Leu26 and the two turns are formed by residues Met40–

Met44 and Asp48–Trp53 [178].

The peptide aptamer chosen in this work being formed of the residues 12-26, it contains the

residues associated with the amphipathic helix which might be considered to explain the

complete inhibition of the electron transfer observed on Figure 3-12. To better understand this

specific behaviour of the monolayer exclusively composed of peptide, we will investigate its

interfacial behaviour by microscopy means in parallel with the mixed layers later in this work.

74

Chapter 4 Elaboration of a p53 peptide-based transducer

for the detection of the protein MDM2

75

Chapter 4. Elaboration of a p53 peptide-based transducer


4.1 Principle of the detection method

In the previous sections, we presented the immobilisation of self-assembled monolayers of

the p53 peptide aptamer mixed with MCB and briefly looked at the behaviour of a layer

exclusively composed of peptide in order to electrochemically characterise and optimise the

interface for the electrochemical biorecognition of the MDM2 target protein by the probes

adsorbed on the gold electrode. We will now present the results associated with the detection

occurring at the electrochemical interface.

The detection of a biorecognition event by electrochemical means can proceed according

to various methods. In this work we propose to use a label-free technique in which the aptamer

probe is not labelled. Since the formation of the p53-MDM2 peptide-protein system will induce

a modification of the electrical double layer, we decided to use the electrochemical impedance

spectroscopy in the presence of the redox couple [Fe(CN)6]3-/4- as detection method. This

technique is widely used in the biosensing field since the electron transfer of the redox couple

is influenced both by a variation in charge at the interface but also by a modification of the

accessibility of the surface. Indeed, it has been shown, in our lab [79], that upon hybridisation,

the charge transfer resistance evolved in opposite ways depending on the type of probe selected,

hairpin DNA or linear DNA. For linear DNA, the hybridisation led to a higher hindrance at the

electrode and the charge transfer resistance increased whereas for hairpin DNA, the

disappearance of the DNA loop upon hybridisation favoured the access of the redox marker to

the electrode inducing a decrease of the charge transfer resistance. The variation of electron

transfer resistance will allow us to compare the interfacial behaviour, associated with the

various procedures of immobilisation considered, as we have seen in section 3.4 that the

electrochemical behaviour of the redox complex is tremendously affected by the adsorption

procedure considered.

Practically, a Nyquist plot of the electrode modified by the aptamer probe mixed or unmixed

with MCB is recorded in a cell containing a 50 mM phosphate buffer at pH 7.4 in the presence

of 5 10-3 M [Fe(CN)6]3- and 5 10-3 M [Fe(CN)6]

4-. The impedance spectrum is recorded at

+0.2 V in a frequency range between 10 mHz and 50 kHz for a monolayer exclusively

composed of peptide and 350 mHz and 50 kHz for mixed layers with an alternating amplitude



76

of 5 mV. The electrode is then taken out from the electrochemical cell and put into contact with

a 1 M phosphate buffer solution - pH 7.4 containing the MDM2 target for 10 minutes.

Afterwards, the electrode is rinsed with the measuring buffer and replaced in the electrolyte for

impedance measurements.

To deeper characterise the electrochemical interface, the experimental impedance spectra

are confronted to theoretical models of electrical equivalent circuits where the electrochemical

cell is represented by a combination of resistors and capacitors. The equivalent circuits

associated with an electrochemical cell are not unique and their identification for complicated

processes are often very complex.

A frequently used circuit, is the Randles equivalent circuit which models interfacial

electrochemical reactions in presence of semi-infinite linear diffusion of electroactives species

at electrodes. Since the responses are rarely the ideally expected ones the distribution of

reactivity is commonly represented in equivalent electrical circuits as a constant phase element

(CPE). The time-constant dispersion leading to CPE behaviour can arise from surface

heterogeneities such as grain boundaries, crystal faces on a polycrystalline electrode or other

variations in surface properties [163]. Therefore, in the Randles equivalent circuit, the double

layer capacitance Cdl is often replaced by a constant phase element Qdl. The Randles electrical

equivalent circuit Rs(Qdl[RctW]) is presented on Figure 4-1 where Rs represents the resistance

of the solution, Rct the charge transfer resistance which is inversely proportional to the exchange

current density and gives us information about the electron transfer kinetics, Qdl the constant

phase element of the double layer and Zw the Warburg impedance which is related to the semi-

infinite linear diffusion of the redox species to the electrode.

Figure 4-1: Illustration of the Randles electrical equivalent circuit Rs(Qdl[RctW]).

Generally, we favour the selection of a simple equivalent circuit. Indeed, although an increasing

number of parameters allows a better fitting of the experimental data, it often lacks of scientific

meaning as the uncertainties on the fitting parameters are too high (> 30%). The choice of the

Randles equivalent circuit has been motivated by its suitability for systems presenting electron

transfer. Indeed, in the context of this work, we will focus mostly on the charge transfer

resistance Rct associated with the electron transfer of the redox couple [Fe(CN)6]3-/4- at the

electrode. This parameter which is influenced both by the variation of charge at the interface

and the modification of accessibility will allow the detection of the recognition event.

http://en.wikipedia.org/wiki/Exchange_current_density

http://en.wikipedia.org/wiki/Exchange_current_density



77

The impedance of a constant phase element, Qdl, is given by:

𝑍𝑄𝑑𝑙

=1

(𝑖𝜔)𝛼𝑌0 (4.1)

with -1 < α < 1 and Y0 the admittance at ω=1 rad s-1 expressed by:

𝑌0 =

1

|𝑍| (4.2)

So that we have,

𝑌0 =

1

𝑅 𝑓𝑜𝑟 𝛼 = 0 (4.3)

𝑌0 = 𝐶 𝑓𝑜𝑟 𝛼 = 1 (4.4)

Therefore, when α=1, the element represents a purely capacitive behaviour.

When α=1/2, the constant phase element evidences a diffusive behaviour which can be

represented by a Warburg element.



78

4.2 Influence of the contact of the protein MDM2 with the

peptide layer on the charge transfer resistance

Figures 4-2 and 4-3 present the impedance spectra as a Nyquist plot of an Ap-cys-p53/MCB

(2/3) modified electrode, obtained by one-step and two-step adsorption respectively, before and

after contact with the MDM2 target protein.

0 500 1000 1500 2000 2500 3000

0

500

1000

1500

2000

2500

3000 Before MDM2

After 5 nM MDM2

-Z''

/ o

hm

Z' / ohm

Figure 4-2: Impedance spectra in 50 mM phosphate buffer – pH 7.4 in the presence of

5 10-3 M [Fe(CN)6] 3-/4- 1/1 at + 0.20 V of coadsorbed Ap-cys-p53/MCB (2/3) monolayer on

gold electrode before (■) and after (●) 10 minutes contact with 5 nM MDM2.



79

0 1000 2000 3000 4000 5000 6000 7000

0

1000

2000

3000

4000

5000

6000

7000 Before MDM2

After 5 nM MDM2

-Z''

/ o

hm

Z' / ohm


5 10-3 M [Fe(CN)6] 3-/4- 1/1 at + 0.20 V of two-step adsorption Ap-cys-p53/MCB (2/3)

monolayer on gold electrode before (■) and after (●) 10 minutes contact with 5 nM MDM2.

The impedance spectra of a peptide alone modified electrode before and after contact with the

MDM2 target protein are presented on Figure 4-4.

0 500 1000 1500 2000 2500 3000

0

500

1000

1500

2000

2500

3000

Before MDM2

After 5 nM MDM2

-Z''

/ k

oh

m

Z' / kohm


5 10-3 M [Fe(CN)6] 3-/4- 1/1 at + 0.20 V of a Ap-cys-p53 alone monolayer on gold electrode

before (■) and after (●) 10 minutes contact with 5 nM MDM2.



80

The analysis in terms of the Randles electrical equivalent circuit, Rs(Qdl[RctW]), are

summarised in Table 4-1 for the three immobilisation situations before and after interaction

with MDM2. Note that the value of the parameter Rs has been set to its experimental value in

the case of coadsorption since the value resulting from the fitting was negative which has no

physical meaning. Note also that in the absence of any apparent diffusion in the case of a

monolayer exclusively composed of peptide, the Warburg impedance has been set to 0. The

error estimates are calculated by testing several solutions near the best fit. For example, if the

best value for a particular resistor is 100 Ohms, the value is increased and decreased until the

goodness of fit starts to decrease. If 98 and 102 Ohms produces a very similar goodness of fit,

but 97 and 103 Ohms produces a poorer fit, the Error is reported as 2/100 * 100 = 2%. The

errors oscillating between 1 and 10 % supports the suitability of this model.

Table 4-1: Fitting parameters of the electrical equivalent circuit Rs(Qdl[RctW])

(The Error estimates are calculated by testing several solutions near the best fit).

Rs (Ω) Rct (kΩ) ZQdl (Ω) Zw (10-3 Ω)

Y0 (10-6 S sα ) α

Coadsorption

Before

MDM2

42.3 0.69

(±0.01)

0.036 (±0.007) 0.98

(±0.02)

0.229

(±0.006)

After 5 nM

MDM2

34.9 0.86

(±0.02)

0.05 (±0.01) 0.95

(±0.02)

0.225

(±0.005)

Two-step

adsorption

Before

MDM2

410 (±4) 3.74

(±0.04)

0.46

(±0.03)

0.894

(±0.007)

0.195

(±0.005)

After 5 nM

MDM2

405 (±4) 3.51

(±0.04)

0.44

(±0.02)

0.900

(±0.007)

0.206

(±0.006)

Peptide alone

Before

MDM2

196

(±19)

2430

(±220)

0.57 (±0.04) 0.88

(±0.01)

0

After 5 nM

MDM2

189

(±18)

809 (±57) 0.67

(±0.06)

0.86

(±0.01)

0

The Nyquist representations illustrated on Figure 4-2, 4-3 and 4-4 show that the charge transfer

resistance before interaction is highly affected by the adsorption procedure. Indeed, this

parameter is increased by a factor 5 between coadsorption and two-step adsorption and by a

factor 3500 for the peptide alone. Major differences in the electron transfer behaviour of the

[Fe(CN)6]3-/4- redox couple have already been observed in this work on the basis of the cyclic

voltammograms of the various layers presented on Figure 3-12. It has been seen that one-step

coadsorption of peptide and MCB leads to a more reversible behaviour of the electron transfer

compared to a two-step adsorption whereas it is totally inhibited when the peptide is

immobilised alone. AFM measurements have confirmed that the thickness of the layer increases



81

respectively according to the ascending order coadsorption, two-step adsorption, and peptide

alone. The values of Y0 of the Qdl element of the equivalent circuit, which is higher for the

peptide alone compared to both mixed layers indicating a higher capacity of the layer, further

support this hypothesis. Furthermore, chronocoulometric measurements carried out on a

peptide layer in the presence of [Ru(NH3)6]3+ has given a charge density of the adsorbed

complex twice higher than for a two-step adsorption layer.

The analysis of the results, presented on Figure 4-2, 4-3 and 4-4, indicates that the charge

transfer resistance in the presence of a coadsorbed layer of Ap-cys-p53/MCB (2/3) reaches 0.69

kΩ prior to detection. After contact with 5 nM MDM2, a slight increase to 0.86 kΩ is observed.

This higher value is probably related to the interaction between the probe and the MDM2

protein which further hinders the access of the redox couple to the electrode surface. Although

an observable modification of the charge transfer resistance is observed after interaction with a

5 nM solution of MDM2 for a coadsorbed layer, the amplitude of this variation is not enough

to allow the detection of lower concentrations of the peptide.

It can also be observed that the interaction of a two-step adsorbed probe with a 5 nM solution

of MDM2, does not lead to significant modification of the shape of the impedance spectra

shown on Figure 4-3. This is confirmed by the analysis in terms of electrical equivalent circuit

since only a decrease of 6 % (from 3.74 kΩ to 3.51 kΩ) is observed. These results indicate that

the two-step adsorption of the probe is unable to provide a good sensitivity for the detection of

MDM2.

On the contrary, it is impressive to note on Figure 4-4 that the interaction of the MDM2 target

protein with a layer of peptide alone leads to a massive drop in the charge transfer resistance

indicating that the electron transfer is facilitated after interaction. Indeed, the charge transfer

resistance varies from 2430 kΩ before interaction to 810 kΩ after interaction which corresponds

to a 67 % decrease. This variation may be explained by a reorientation of the p53 peptide probe

upon interaction with MDM2. Indeed, in the absence of a diluent thiol, the presence of non-

specific interactions between the peptide probe and the gold surface is very likely. Upon

interaction with MDM2, the peptide probe could take an upright conformation to interact with

the target. The transition from a random structure to a α helix of the p53 transactivation domain

upon interaction with MDM2 has been extensively studied [137, 144]. From these results, the

detection mechanism based on the release of the electron transfer of a layer exclusively

composed of peptide upon interaction with MDM2 seems the most promising. This prospect

which is quite unusual since mixed layers are widely used and promoted in the literature could

lead to many possibilities for the detection of proteins.

Prior to the testing of the Ap-cys-p53 based sensor, we will try to identify whether the

signal modification is really related to the interaction with the target protein. Indeed, the

observed decrease in signal could also be related to the replacement of the peptide probe by the

target protein, which also presents cysteine residues able to form a covalent bond with gold, or,

to the removal of the non-specifically adsorbed peptide upon interaction. In the following



82

section, we will therefore present the behaviour of the MDM2 target protein at a bare gold

electrode in order to validate or not the use of a monolayer exclusively composed of the peptide

probe as a transducer for the detection of MDM2.

4.3 Behaviour of the MDM2 protein on gold and at Ap-

cys-p53 modified gold electrodes

Figure 4-5 shows the reductive desorption curve recorded on a gold electrode that has been

modified with the MDM2 protein overnight. While sweeping from 0.00 V to -0.80 V, the

occurrence of a reduction peak at -0.59 V is observed during the first scan. This indicates that

the protein can covalently bind to gold. Indeed, the protein contains cysteine residues allowing

the formation of a sulphur-gold bond. While scanning back to more positive potentials, a small

oxidation peak appears at -0.51 V. As it has been seen in section 3.2, the smaller intensity of

the oxidation peak compare to the reduction peak can be explained by the diffusion of a large

amount of protein to the bulk upon desorption before the potential allowing a re-adsorption is

reached. Therefore, only the proteins remaining in the vicinity of the gold surface will be re-

adsorbed.

-0.8 -0.6 -0.4 -0.2 0.0

-20

-15

-10

-5

0

5

First cycle

j /

µA

.cm

-2

E vs Ag|AgCl / V

Figure 4-5: Cyclic voltammogram of a polycrystalline gold electrode modified overnight by

MDM2 recorded in 10 mM Tris-HCl-pH 7.4; υ = 50 mV s-1.

Figure 4-6 shows an impedance spectrum of a gold electrode modified overnight by MDM2

recorded in the same conditions as earlier. Contrary to the results obtained on a one component

Ap-cys-p53 layer, the charge transfer resistance, obtained by analysing the data according to a

Randles equivalent circuit, reaches 3.2 kΩ, a value 80 times lower than that obtained for the

peptide layer indicating a much faster electron transfer process. As it has been shown on Figure



83

4-4, the charge transfer resistance strongly decreased after the recognition event. This might

indicate that the protein MDM2 removes the peptide layer. However, the charge transfer

resistance remains 17 times higher, after interaction, than that of a gold electrode modified by

the MDM2 protein. These observations indicate that the variation observed in the charge

transfer resistance of an Ap-cys-p53 alone monolayer upon contact with MDM2 cannot be

simply explained by the replacement of the peptide probe by MDM2.

0 1000 2000 3000 4000 5000 6000 7000

0

1000

2000

3000

4000

5000

6000

7000

-Z''

/ ohm

Z' / ohm

Figure 4-6: Impedance spectrum of a MDM2 monolayer on gold electrode in 0.05 M

phosphate buffer – pH 7.4 in the presence of 5 10-3 M [Fe(CN)6] 3-/4- 1/1 at + 0.20 V.

Reductive desorption experiments, whose associated voltammetric curves are presented on

Figure 4-7, carried out in the presence of an Ap-cys-p53 layer (a) and on an Ap-cys-p53 layer

after 10 minutes of contact with a 5 nM MDM2 solution (b), have confirmed the remaining

presence of the peptide probe after the recognition event. Indeed, by sweeping the potential

from 0.00V to -0.80 V, a first peak appears at -0.59 V followed by a second small peak at -

0.72V. The comparison with Figure 4-5 and Figure 4-7(b) allows the association of the first

peak with MDM2 and of the second with the peptide probe. These data give direct evidence for

the adsorption of MDM2 at the peptide modified electrode and, suggest that the peptide remains

present at the surface after interaction.



84

-0.8 -0.6 -0.4 -0.2 0.0-10

-8

-6

-4

-2

0

-0.8 -0.6 -0.4 -0.2 0.0

-8

-4

0(a)

j /

µA

.cm

-2

E vs Ag|AgCl / V

(b)

j /

µA

.cm

-2

E vs Ag|AgCl / V

Figure 4-7: Linear sweep voltammetry of a Ap-cys-p53 modified gold electrode before (a)

and after (b) 10 minutes contact with 5 nM MDM2 in 10 mM Tris-HCl – pH 7.4,

υ = 50 mV s-1.

To further support this statement, we carried an experiment where the Ap-cys-p53 probe

layer was replaced by its fluorescently-labelled equivalent. To evidence the absence of

replacement of our peptide probe by the MDM2, we proceeded to the reductive desorption of

the Ap-cys-p53-fluo layer after 10 minutes contact with 5 nM MDM2. Electrochemical

impedance measurements were similar for fluorescently labelled or not probes.

Figure 4-8 presents the capacitance and mean fluorescence on a full image as a function of the

applied potential. Prior to the desorption of the layer, no fluorescence is observed since the

adsorbed labelled biomolecules undergo quenching by the gold surface. Upon desorption, the

quenching is no longer effective and a fluorescence signal is recorded. This phenomenon will

be described in further details later in this work. Although the increase in capacitance,

witnessing the desorption of the adsorbed biomolecules, can be correlated to the presence at the

electrode of both MDM2 and p53, the observed increase in fluorescence observed can only be

due to the fluorescently labelled p53 probe. A first increase appears between -0.40 V and -0.70

V and can be associated to the two small peaks observed on Figure 4-7(a) corresponding to the

desorption of the peptide. This indicates that the probe remains on the gold surface after the



85

interaction with the target protein. The second increase appearing at about -1.25 V is probably

due to the reduction of the electrolyte.

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.20

1

2

3

4

5

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2

1000

1100

1200

1300

1400

1500 (b)

C /

µF

E vs Ag|AgCl / V

(a)

Mea

n f

luore

scen

ce i

nte

nsi

ty /

a.u

.

E vs Ag|AgCl / V

Figure 4-8: Capacitance-potential (a) and mean fluorescence-potential (b) curves of a Ap-cys-

p53-fluo modified electrode after 10 minutes contact with 5 nM MDM2 recorded in 10 mM

phosphate buffer-pH 7.4.



86

4.4 Quartz crystal microbalance measurements

As electrochemical measurements give indirect evidence of the interaction, the interaction

process has also been followed by a non-electrochemical technique, namely by quartz crystal

microbalance measurements, to validate the strategy presented above. Indeed, quartz resonators

are often used as mass sensors, to monitor molecular recognition events in biological sensors,

since it is able to transform a mass intake of an analyte into an electrical signal. It has, for

example, been used to monitor hybridisation events, lipid-protein interactions at lipid

membranes, DNA aptamers-protein interactions or the adsorption of proteins at functionalised

interfaces [179, 180, 181, 182]. Therefore, QCM appears as a suitable technique for the

validation of our detection strategy.

We performed measurements on Ap-cys-p53 modified gold coated quartz resonators to

measure how the mass load is influenced upon contact with MDM2 but also with three non-

specific proteins as negative control namely, bovine serum albumin, fibrinogen and cytochrome

c. Table 4-2 summarises some information relative to those proteins.

Table 4-2: Some physical properties of MDM2, BSA, fibrinogen and cytochrome c.

MDM2 BSA Fibrinogen Cytochrome c

Molecular weight 58 kDa 66 kDa 341 kDa 12 kDa

Isoelectric pH 4.6 4.7 5.4-5.5 10.0 - 10.5

Charge at pH 7.4 - - - +

As can be seen from Table 4-2, the proteins selected as negative controls vary in terms of

isoelectric pH and charge, and in terms of molecular weight. Albumin from bovine serum has

been selected because it presents a similar molecular weight and isoelectric pH compared to

MDM2. Besides, this protein is present in large amount in serum which makes it a major

potential interfering agent. BSA is also often used as a model system for protein adsorption and

is even used as an antifouling agent in the preparation of ELISA devices [183]. Fibrinogen was

selected because of its bigger size and its ability to adsorb on various substrates upon blood

contact which makes it a key factor in the determination of heamocompatibility of a material

[184]. Finally, cytochrome c was selected for its high isoelectric pH which makes it positively

charged at the working pH of 7.4. Indeed, since the peptide is negatively charged, the use of a

positively charged protein as negative control will allow to study whether the charge of the

protein influence the electrochemical impedance measured signal.

A 20 µM Ap-cys-p53 solution was immobilised overnight on the gold crystal in 1 M phosphate

buffer – pH 7.4. The crystal was then placed in a flow cell and rinsed by the same buffer before



87

injection of a 5 nM protein solution. Once the whole solution has gone through the flow cell,

the crystal is rinsed again with the buffer. Figure 4-9 presents the data obtained with both

MDM2 and negative control proteins.

0 250 500 750 1000 1250 1500 1750

-0.15

-0.10

-0.05

0.00

0.05

0.10

1 M Phosphate

Buffer

Injection

Fibrinogen

m

/ µ

g

t / s

MDM2

BSA

Fibrinogen

Cytochrome c

Injection

MDM2

1 M Phosphate

Buffer

Figure 4-9: Variation in mass of the Ap-cys-p53 modified gold coated quartz crystal (10

MHz) after contact with a 5 nM solution of MDM2 ( ), BSA ( ), Fibrinogen ( ) or

Cytochrome c ( ) in 1 M phosphate buffer – pH 7.4.

It can be seen from Figure 4-9 that only MDM2 induces an increase in mass whereas BSA,

fibrinogen and cytochrome c induce a mass decrease. The increase in mass upon contact with

MDM2 is easy to understand since it is the expected behaviour upon recognition. However, the

behaviour observed for negative controls has to be looked into further details since non-specific

adsorption of these proteins should have led to the opposite effect.

It is interesting to note that upon rinsing with the buffer after contact with the negative controls,

the mass tends to its initial value indicating that the decrease does not actually correspond to a

mass loss. This behaviour is probably due to the change of solution that occurs upon injection

of the solution containing the negative control protein and might be related to a viscosity effect.

Indeed, through the preparation of the protein solution a 5 % dilution of the 1 M phosphate

buffer is introduced since proteins are initially dissolved in 20 mM Tris-HCl. This dilution is

presumed to sufficiently modify the viscosity of our solution so that a modification of the

frequency appears upon injection of the protein solution.

Kanazawa and Gordon predicted the influence of the viscosity of the media on the frequency

of vibration of the quartz [185, 186]. The variation in frequency is given by equation 4.5:



88

∆𝑓 = −𝑓03/2

√𝜂𝑠𝜌𝑠

𝜋𝜇𝑞𝜌𝑞 (4.5)

with f0 the fundamental frequency of the free dry crystal, ηs the kinematic viscosity of the

solution; ρs the volumetric mass density of the solution; µq the shear modulus of the quartz and

ρq volumetric mass density of the quartz. This relation was experimentally verified by

Bruckenstein and Shay by varying proportions in glycerol-water mixtures [187].

Figure 4-10 presents QCM data obtained upon injection of a 5 % diluted solution of 1 M

phosphate buffer. A decrease of similar amplitude to that obtained for cytochrome c and

fibrinogen is observed confirming the hypothesis of a viscosity effect.

0 200 400 600 800 1000 1200 1400 1600-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0.02 1 M phosphate buffer

m

/ µ

g

t / s

5 % diluted

1 M phosphate buffer

Figure 4-10: Influence of the viscosity of the media on the variation in mass measured by

quartz crystal microbalance.

All four protein solutions presenting the same dilution factor, as they were prepared according

to a very same procedure, it appears that the change in mass observed for cyctochrome c and

fibrinogen originates from the variation in viscosity rather than from an effect of protein

interaction since it presents a similar variation as that recorded by injection of a diluted solution.

Besides, we assume that the smaller decrease observed for BSA might be due to some non-

specific adsorption of this protein that would be hidden by the influence of the viscosity. This

is not surprising since it presents similar isolelectric pH and molecular weight to MDM2. It

appears that only the interaction of our target protein, MDM2 with the probe, is sufficient

enough to overcome the viscosity effect.



89

From the data presented on Figure 4-9, an increase in mass of about 0.05 µg is recorded

indicating an adsorption of 2.5 1012 molecules cm-2, corresponding to a projected area per

MDM2 of 4000 Å². It is important to keep in mind that this value is probably underestimated

since the variation in mass obtained by QCM measurements is affected by the viscosity effect.

A variation in mass of 0.15 µg is probably more accurate and corresponds to 7.6 1012

molecules cm-2 and a projected area of 1300 Å² per MDM2.

From chronocoulometric measurements carried out on a layer exclusively composed of

Ap-cys-p53, in the presence of [Ru(NH3)6]3+ complex, a density of adsorbed charge of 12 µC

cm-2 has been recorded. Assuming a one-to-one ratio between the complex and the peptide

probe, a density of adsorbed probe of 7.5 1013 molecules cm-2 has been evaluated. From these

data, considering a variation in mass corrected by the viscosity effect of 0.15 µg, a ratio of 1

MDM2 protein for 10 Ap-cys-p53 probes has been reached.



90

4.5 Analytical performance

4.5.1 Impact of the concentration of MDM2 on the signal

In the previous section, we showed that incubation of a monolayer exclusively composed

of the p53 aptamer with a solution containing the MDM2 protein led to significant modification

of the charge transfer resistance. To evaluate whether this strategy can be implemented for

biosensing, we have to assess its analytical performance.

To evaluate the recognition properties of the system, in terms of sensitivity and selectivity,

we have performed electrochemical impedance spectroscopy on Ap-cys-p53 aptamer modified

gold electrodes before and after exposing the electrode to various MDM2 concentrations. As

shown on Figure 4-11, the electron transfer which is highly inhibited before interaction, already

partially recovers at concentrations of protein as low as 10 pM.

0 500 1000 1500 2000 2500 3000

0

500

1000

1500

2000

2500

3000

0 nM

0.005 nM

0.01 nM

0.02 nM

0.05 nM

0.07 nM

0.1 nM

0.2 nM

0.5 nM

0.7 nM

1 nM

2 nM

5 nM

7 nM

10 nM

-Z''

/ kohm

Z' / kohm

Figure 4-11: Nyquist plots of impedance data recorded before (■) and after 10 minutes

contact with various concentration of MDM2 in 50 mM phosphate buffer- pH 7.4 in the

presence of 5 mM [Fe(CN)6]3-/4- 1/1 at + 0.20 V.

The changes of resistance are calculated according to equation 4.6:

∆𝑅𝑐𝑡 (%) =

𝑅𝑐𝑡𝑝53+𝑀𝐷𝑀2 − 𝑅𝑐𝑡

𝑝53

𝑅𝑐𝑡𝑝53 × 100 (4.6)



91

with 𝑅𝑐𝑡𝑝53 the charge transfer resistance before recognition and 𝑅𝑐𝑡

𝑝53+𝑀𝐷𝑀2 after incubation in

the MDM2 solution.

The charge transfer resistance decreases as the concentration in MDM2 increases until reaching

a roughly constant value above 1 nM (Figure 4-12).

0 2000 4000 6000 8000 10000

-80

-60

-40

-20

0

R

ct /

%

[MDM2] / pM

Figure 4-12: Calibration plot showing the change in charge transfer resistance as a function of

different concentrations of MDM2 (n=4).

Figure 4-13: Modification of the charge transfer resistance (∆Rct) with the log of MDM2

concentration (n=4).

1.0 1.5 2.0 2.5 3.0-90

-80

-70

-60

-50

-40

-30

-20

-10

0

R

ct /

%

log[MDM2] / pM



92

The analytical sensitivity, measuring the ability of the method to discriminate small variations

in the analyte concentration, is given by:

𝛾 = 𝑚/𝑠𝑆 (4.7)

with γ the analytical sensitivity, m the slope of the calibration curve and ss, the standard

deviation of the measurement.

A linear relationship between ∆Rct and logarithmic value of MDM2 concentration was found in

the range of 10 to 1000 pM (0.58 ng mL-1 to 58 ng/mL) as shown on Figure 4-13. This semi-

logarithmic representation is typical of the calibration curves associated with the binding

between a protein and a ligand. This representation leads to a characteristic S-shaped curve

which central part is linear and allows quantification. At a concentration of 100 pM, which is

the one presenting the highest standard deviation, an analytical sensitivity of 2 has been

calculated.

4.5.2 Negative controls

To confirm that the modifications in charge transfer resistance of the impedance curves

arise from the specific interaction between Ap-cys-p53 and MDM2, and to evaluate the

selectivity of the recognition, control experiments were performed. These measurements will

allow the determination of a realistic limit of detection of the method. To do so, the same non-

specific proteins as for quartz microbalance measurements have been used: bovine serum

albumin, fibrinogen and cytochrome c.

Figure 4-14 presents the impedance data obtained before and after interaction of the probe with

(a) BSA, (b) fibrinogen and (c) cytochrome c. Impedance spectra were recorded before and

after 10 minutes contact with 5 nM solutions of each of the three non-specific proteins.



93

0 50 100 150 200 250 300 350

0

50

100

150

200

250

300

350 Before BSA

5 nM BSA

-Z''

/kohm

Z' / kohm

(a)

0 50 100 150 200 250 300 350

0

50

100

150

200

250

300

350 Before fibrinogen

5 nM fibrinogen

-Z''

/ kohm

Z' / kohm

(b)



94

0 500 1000 1500 2000 2500 3000 3500

0

500

1000

1500

2000

2500

3000

3500

-Z''

/ kohm

Z' / kohm

Before cytochrome c

5 nM cytochrome c(c)

Figure 4-14: Impedance spectra of a Ap-cys-p53 modified electrode before (■) and after (●)

10 minutes contact with 5 nM BSA (a), Fibrinogen (b), Cytochrome c (c), measured in 0.05

M phosphate buffer – pH 7.4 – 5 10-3 M [Fe(CN)6] 3-/4- 1/1.

From the control data presented on Figure 4-14, we observe a small decrease

(ΔRct = 15.4 ± 0.8 % ; n=3) of the charge transfer resistance after contact with solutions of the

three non-specific proteins compared to that obtain for a same concentration in MDM2. This

supports the assumption according to which the reduction in charge transfer resistance recorded

upon exposure of the Ap-cys-p53 modified gold electrode to various concentrations of MDM2

is related to a specific binding of the protein to its probe. From these data, we can extract the

limit of detection (LOD), within a confidence limit of 99.7 %, corresponding to the lowest

quantity of a substance that can be distinguished from the absence of that substance, according

to equation 4.8:

𝐿𝑂𝐷 = 𝑆�̅�𝑐 + 3𝑠𝑛𝑐 (4.8)

where 𝑆�̅�𝑐 is the mean of the variation of charge transfer resistance in percentage of the negative

control signals and snc its standard deviation. The limit of detection has been based on the

negative controls rather than the blank because of its higher relevance. Indeed, contrary to non-

specific proteins, no interfering signal is expected upon contact with buffer solution.

The limit of quantification (LOQ) can also be determined according to the relation:

𝐿𝑂𝑄 = 𝑆�̅�𝑐 + 10𝑠𝑛𝑐 (4.9)



95

A limit of detection of 12 pM, corresponding to 0.69 ng mL-1 and a limit of quantification of

19 pM, corresponding to 1.08 ng mL-1 have been obtained.

ELISA (enzyme-linked immunosorbent assay) is a common technique used to identify the

presence of biomolecules as antibodies, antigen or protein in a sample. The information

gathered on performances offered by various ELISA kits indicate a detection range being, at

best, between 0.156 ng mL-1 and 20 ng mL-1 and a limit of detection reaching 0.053 ng mL-1

[188]. The detection range of 1.08 to 58 ng mL-1 and LOD of 0.69 ng mL-1 obtained with this

transducer, indicates that the impedimetric detection, based on the immobilisation of a peptide

aptamer, elaborated in this work, can compete with the performance of an ELISA kit.

Furthermore, the detection is much faster. Indeed, an ELISA analysis takes about 45 minutes

against 15 minutes in our case. The new strategy, considered in this work, appears very

promising and opens a wide range of possibilities in the sensing of other proteins since the

technique is based on the immobilisation of a peptide allowing the interaction with a target

protein.

96

Chapter 5 Fluorescence microscopy study of self-

assembled monolayers of the peptide aptamer probe on gold

97

Chapter 5. Fluorescence microscopy study of self-

assembled monolayers of the peptide aptamer probe on

gold

It has been demonstrated, in the previous chapters, that different adsorption procedures

lead to different electrochemical behaviours both in the presence of positively and negatively

charged redox marker. More surprisingly, it has also been shown that the most suitable

transducer, with respect to the considered strategy, is the one exclusively composed of the

peptide.

As electrochemistry only provides average information on what is happening at the electrode

surface, we propose, in the following section, to examine, at a localised scale, the organisation

of the layers according to the various adsorption procedures considered in this work. To do so,

in situ epifluorescence microscopy under potential control, a unique technique able to give

simultaneously localised and average information on the monolayers characteristics, will be

considered.

5.1 Fluorescence Spectroscopy

5.1.1 General principles

The fluorescence phenomenon corresponds to the re-emission of an absorbed photon

without modification of the spin of the electron as the transition occurs between a singlet excited

state and a singlet ground-state. The emission rates of fluorescence are about 108 s-1,

corresponding to a fluorescence lifetime of 10 ns. A Jablonski diagram represents the various

molecular processes that can occur after the absorption of photons as shown on Figure 5-1.



98

Figure 5-1: A Jablonski diagram showing the transitions between energy levels [189].

It can be seen on the Jablonski diagram that the energy of emission is lower than the energy

of absorption meaning that fluorescence occurs at longer wavelengths. This phenomenon

originates in the fact that a molecule can be excited at any vibrational state. In solution, this

excess of energy is lost through collisions between the excited molecules and those composing

the solvent. Therefore, a transition in fluorescence always originates from the lower vibrational

level of the excited state. A superimposition in the absorption and emission spectra is only

observed in the range of wavelengths corresponding to the lines of resonance which are

associated with a transition between the lowest vibrational levels of the excited and ground

states.

For most fluorophores, the emission spectrum is the mirror image of So → S1 absorption,

not of the total absorption spectrum. The symmetry is caused by the fact that transitions between

same vibrational energy levels are involved in both the absorption and emission processes. For

most fluorophores, these energy levels are not significantly altered by the different electronic

distributions of So and S1. Indeed, if the peaks associated with the absorption of a fluorophore

are due to transitions from the 0th vibrational state of the ground state So to higher vibrational

levels of the excited state S1, the emission can return to any of the vibrational state of the ground

state. Furthermore, the vibrational energy difference being similar in both So and S1, the

vibrational energy difference is the same in both emission and absorption spectra. All the

transitions occur without change in the position of the nuclei according to the Franck-Condon

principle, if the probability of a transition between the 0th and the 1st vibrational levels is largest

in absorption, the same is true in emission. This mirror-image rule is illustrated on Figure 5-2.



99

Figure 5-2: Mirror-image rule [190].

5.1.2 Fluorescence quenching

An excited molecule can return to the ground state through various successive processes.

Knowing that the excited molecules not only relax to the ground state through radiative

processes but also via non radiative processes, two major parameters are considered to

characterise a fluorophore, the fluorescence lifetime and the quantum yield.

The fluorescence quantum yield is defined as the fraction of excited molecules that return

to the ground state S0 with emission of fluorescence photons. In other words, the fluorescence

quantum yield is the ratio of the number of emitted photons (over the whole duration of the

decay) to the number of absorbed photons and is given by:

Φ =

𝑘𝑟

𝑘𝑟 + 𝑘𝑛𝑟 (5.1)

where kr and knr are respectively the rate constant for radiative deactivation of the fluorophore

and its rate constant of non radiative decay to S0.

The lifetime is defined as the time spent by a molecule in the excited state before it returns

to the ground state and is given by:

𝜏 =

1

𝑘𝑟 + 𝑘𝑛𝑟 (5.2)



100

In the absence of non radiative processes, the natural or intrinsic lifetime of the fluorophore

is expressed by:

𝜏0 =

1

𝑘𝑟 (5.3)

The most favourable path being the one that minimises the lifetime of the excited state, if

the kinetics of the non radiative process is favoured, the intensity of fluorescence will be

minimised or even suppressed. This decrease in fluorescence intensity is called “quenching”.

Among the non radiative processes, the internal conversion describes intermolecular processes

allowing a molecule to return to a lower electronic state without light emission. This

phenomenon occurs when two energy levels are close enough to present vibrational levels of

same energy. In general, this process is more likely than fluorescence from a higher excited

state. In the Jablonski diagram presented on Figure 5-1, this process is illustrated between the

vibrational levels of the excited states S2 and S1.

Another quenching mechanism, which occurs from the first singlet excited state S1 to the first

triplet state T1, is intersystem crossing, possible thanks to spin-orbit coupling. The efficiency

of this coupling varies with the fourth power of the atomic number, so that intersystem crossing

is favoured by the presence of a heavy atom.

A third process is the collisional quenching which occurs when an excited molecule is

deactivated upon contact with the solvent or other solutes. Many molecules can act as

collisional quenchers such as oxygen, halogens, and amines. The decrease in fluorescence

associated with this process is well described by the Stern-Volmer equation:

𝛷0

𝛷=

𝐼0

𝐼= 1 + 𝐾𝑆𝑉[𝑄] = 1 + 𝑘𝑞𝜏[𝑄] (5.4)

where I and I0 are the steady-state fluorescence intensities in presence and absence of quencher,

KSV is the Stern-Volmer quenching constant which expresses the sensitivity of the fluorophore

to a quencher, kq is the bimolecular quenching constant, τ lifetime in absence of quencher and

[Q] the quencher concentration. It is interesting to note that KSV-1 is the concentration at which

50 % of the intensity is quenched.



101

5.1.3 Förster Resonance Energy Transfer

In the context of this work, we will take advantage of the quenching of the fluorescence of

excited molecules placed in the vicinity of a metal surface to study the assembly of fluorescently

labelled peptide molecules on gold. This property of quenching by a metal is often associated

with resonance energy transfer.

Whereas quenching results in the dissipation of the energy as heat, resonance energy transfer

(RET) decreases the intensity of the donor (fluorophore) and transfers the energy to the

acceptor, which can be fluorescent or not, through dipole-dipole coupling. This phenomenon

occurs when the emission spectrum of the donor overlaps with the absorption spectrum of the

acceptor (A) so that several vibronic transitions in the donor (D) have practically the same

energy as the corresponding transitions in the acceptor. Such transitions are couple, i.e. are in

resonance. During a RET process, the electron in the excited state of the donor returns to the

ground state while simultaneously an electron in the acceptor goes from the ground state into

an excited state orbital. There is no emission of light by the donor. If the acceptor is fluorescent,

light will be emitted by the acceptor, if not the energy will be released as heat. Unlike collisional

quenching, RET does not require a contact between the donor and acceptor. The distance at

which RET reaches 50 % of efficiency is called the Förster distance. The RET efficiency (ΦT)

depends on the distance between the donor and acceptor and the extent of spectral overlap, and

is given by:

𝛷𝑇 =

𝑘𝑇(𝑟)

1𝜏𝐷

+ 𝑘𝑇(𝑟)

(5.5)

with the rate constant for transfer between a donor and an acceptor at distance r,

𝑘𝑇(𝑟) =

1

𝜏𝐷(

𝑅0

𝑟)

6

(5.6)

where τD is the excited-state lifetime of the donor in the absence of transfer, r is the center-to-

center distance between D and A, and R0 is the Förster critical radius (distance at which transfer

and spontaneous decay of the excited donor are equally probable, i.e. 𝑘𝑇 =1

𝜏𝐷).

Therefore, for a single donor-acceptor pair,

𝛷𝑇 =

𝑅06

𝑟6 + 𝑅06 (5.7)



102

It is interesting to note that RET is efficient over long distances (up to 10 nm) whereas

quenching is efficient at very short distance (few Angströms). Therefore, RET has been

commonly used to evaluate the distance between sites in proteins but also between two proteins

[191, 192].

5.1.4 Fluorescence near metal surface

When an excited fluorescent molecule is placed in the vicinity of a metal surface, its

lifetime can be substantially altered due to the reflection and absorption at the surface. It has

been shown that for large distances from the metal surface, the fluorescence lifetime oscillates

as a function of the distance while for small distances, the lifetime tends towards zero [193]. In

this section, we will consider the phenomenon taking place in the vicinity of the surface that

finds its origin in the non radiative transfer of energy from the excited molecule to the metal.

The emitting molecule acts as an oscillating dipole near a partially absorbing and partially

reflecting surface which can be a very thin metal film as described by Kuhn [194].

This scenario will give electrochemistry a useful tool to study the organisation resulting from

the assembly process of thiolated fluorescent biomolecules on gold electrodes. Indeed, the

immobilised fluorescent biomolecules will act as emitter whereas the gold substrate will play

the role of acceptor.

The transfer of energy from an emitter molecule D to an acceptor A can be treated by

calculating the rate of absorption by the acceptor kT in the field of the emitter to estimate how

the luminescence of the emitter D is quenched by the acceptor A. The amount of quenched

luminescence of the emitter can be expressed as the ratio between the quantum yield of the

emitter in the absence of an acceptor, Φ0 and the quantum yield of the emitter when the acceptor

is located at a distance r from it, Φr.

This relation in terms of the power loss of D, in the absence of A (P0) and the power absorbed

by A in its presence (P), is written as:

𝛷0

𝛷𝑟= 1 +

𝑃

𝑃0 (5.8)

with, the power radiated by the dipole emitter is:

𝑃0 = µ02𝜔4𝑛/3𝛷0𝑐³ (5.9)



103

with µ0 the amplitude of the dipole moment, ω the angular velocity, n the refractive index of

the medium, c the speed of light and 𝛷0 the quantum yield of the dipole emittor in the absence

of acceptor.

If we look at a thin metal absorbing layer of absorbance A for a light of angular velocity

ω, the average rate of energy absorption of an area dxdy of the metal, when irradiated with light

of intensity I striking area with normal incidence, we have:

𝑑𝑃 = 𝐴𝐼𝑑𝑥𝑑𝑦 = 𝐴 (𝑐𝑛

8𝜋) 𝐹0

2𝑑𝑥𝑑𝑦 (5.10)

with Fo, the amplitude of the electric field.

Let us now replace the incident light beam by the field of a dipole D at a point x,y of the

absorbing metal layer, oscillating in the z direction, perpendicular to the metal absorbing layer

and at a distance r above it as represented on Figure 5-3.

Figure 5-3: Oscillating dipole D at a distance r from an absorbing metal layer.

Assuming that the layer only interacts with the component of the field oscillating in the plane

of the layer with an amplitude F0, we obtain the power absorbed by the acceptor:

𝑃 = 𝐴

1

16 (𝑐𝑛)𝜇0

2 (𝜔4

𝑐4) [

3

2 (

𝜆

2𝜋𝑛𝑟)

4

+ (𝜆

2𝜋𝑛𝑟)

2

+ 1] (5.11)



104

When the acceptor is located at r<<λ, the last term, which is predominant in the radiation field

region (r>>λ), can be neglected and by replacing (5.8) and (5.9) in (5.11) we have:

𝛷

𝛷𝑟= 1 + (

𝑟0

𝑟)

4

+ 𝜂 (𝑟0

𝑟)

2

(5.12)

With

𝑟0 = 𝛼 (𝜆

𝑛) (𝐴𝛷)

1

4 ; (5.13)

𝛼 = (

1

4𝜋) (

9

2)

1

4 ; (5.14)

𝜂 = (

√2

4) (𝐴𝛷)

12 (5.15)

It has been shown that the relation can be reduced at the first two terms by considering a dipole

in the x direction (parallel to the acceptor metal) with an acceptor metal interacting only with

F0 (amplitude of field component oscillating in the plane of the metal). We have then:

𝛷

𝛷𝑟= 1 + (

𝑟0

𝑟)

4

(5.16)

with r0, the Förster distance.

Figure 5-4 represents the evolution of the quenching of the luminescence of a dipole emitter by

a thin metal layer as a function of the distance for various values of the absorbance of the metal.

The values selected for the quantum yield and excitation wavelength corresponds to the

fluorescein fluorophore used in this work. A refractive index of 1.33 has been selected since all

experiments were performed in aqueous solutions.



105

0 50 100 150 200 250 3000.0

0.2

0.4

0.6

0.8

1.0

r /

0

r / nm

A= 0.1

A= 0.25

A= 0.5

A= 0.75

A= 1

Figure 5-4: Evolution of the amount of quenched luminescence (Φr/Φ0) of a dipole emitter by

a thin metal layer acceptor as a function of the distance for various values of the absorbance

of the metal with Φ0= 0.9; λ= 521 nm; n=1.

As can be seen from Figure 5-4, the Förster distance, at which half of the fluorescence is

quenched, is of the order of a few tens of nm. This confirms that, in the context of this work,

the quenching of the fluorescence by a metal can be used as a complementary tool to study how

different procedures of immobilisation influence the organisation of the resulting self-

assembled monolayers. As the quenching by the metal operates at a distance similar to the size

of a biomolecule, a minimum of fluorescence should be observed as long as the biomolecule is

adsorbed on the surface. It has been shown by Rant et al., that upon application of a bias

potential, the orientation of DNA oligonucleotides immobilised on gold could be modulated

[195]. They evidenced that, by consecutively applying positive and negative potential to the

modified interface, respective attraction and repulsion of DNA oligonucleotides towards the

surface changed their orientation from a down to upright position allowing the study of the

interface. This flipping behaviour was followed by fluorescence measurements since an

increase in intensity was recorded while oligonucleotides stood up straight. As a result, the

study of the interface can also be performed via the reductive desorption of the biomolecular

layer under potential control. Indeed, upon desorption of the biomolecules, quenching will be

less effective, so that information can be obtained regarding the adsorbed layer [99, 104, 176,

196].

Electrochemical measurements presented in the first part of this work, such as reductive

desorption of the layer give us an insight of what is happening at the µm scale. The



106

concentration-distance profile of the species during desorption can be described in terms of a

semi-infinite linear diffusion process. By resolving the Fick’s equations, the evolution of the

concentration as a function of time and distance from the electrode can be expressed by

equation 5.17:

𝑐(𝑟, 𝑡) =

𝛤

√𝜋𝐷𝑡 𝑒

−𝑟²4𝐷𝑡 (5.17)

With Γ the total surface concentration in thiols, D the diffusion coefficient of the desorbed

species, t the time and r the distance from the electrode surface.

Figure 5-5 presents the concentration profile as a function of the distance from the electrode for

various times considering a fast desorption. The value selected for the diffusion coefficient and

total surface concentration respectively are typical values for thiol molecules in aqueous media

and densely packed thiol monolayers.

0 100 200 300 4000

20

40

60

80

100

Conce

ntr

ati

on

/ µ

M

r / µm

5 s

10 s

20 s

50 s

100 s

500 s

800 s

Figure 5-5: Concentration profiles of a desorption at different time scales based on the

resolution of the Fick’s equations with D = 2.10-6 cm² s-1; Γ=5.10-10 mol/cm².

As can be seen on Figure 5-5, mass transport influences electrochemical data at a µm scale

from the surface, whereas the quenching properties of the metal are impacted at a nm scale from

the metal surface. Therefore, we propose to couple fluorescence microscopy and

electrochemical measurements to study in more details how the immobilisation procedure

impacts the organisation of the probe layer. Furthermore electrochemistry gives us average

information on the phenomenon occuring at the electrode surface, whereas fluorescence

microscopy will allow us to obtain some local information.



107

In order to proceed to the experiments, the peptide probe sequence has been modified by a

fluorescent marker.

5.1.5 Fluorescence Microscopy

In fluorescence microscopy, the wavelength is selected through the use of optical filters. In

this work, the microscope will present an epifluorescence configuration, meaning that both the

excitation and emission beams are passing through the microscope objective. To observe the

fluorescence, a set of filters represented on Figure 5-6 is required. It is composed of three

combined categories of filters; excitation filters, barrier or emission filter and dichromatic

beamsplitter which spectral properties are represented respectively by the blue, red and green

curve on Figure 5-7.

Figure 5-6: Illustration of a filter cube [197].

A range of wavelengths is selected by the excitation filter from a broadband source to excite

the sample. The resulting emission is transmitted by the emission filter which rejects the

excitation wavelengths. The dichroic mirror reflects the excitation wavelength into the objective

and transmits the emission to allow it to reach the eyepiece or the detector. Figure 5-7 presents

the spectral characteristics associated with the filter cube adapted to the 5-carboxyfluorescein

fluorophore used in this work. The excitation filter, only allows the transmission of selected

wavelengths from the lamp to the specimen. In the case of the 5-carboxyfluorescein fluorophore

used in this work, excitation is allowed for wavelengths from 450 to 490 nm as shown by the

blue curve. The emission filter, playing the role of barrier, is designed to absorb the excitation

wavelengths and only allows the selected emission wavelengths, in this case from 515 nm, to

go towards the detector as represented by the red curve. The dichroic mirror reflects the

excitation wavelengths and passes emission ones from 505 nm as presented by the green curve.

The 5-carboxyfluorescein spectra are shown behind the filters curves with maximum

wavelengths of absorption and emission respectively at 492 nm and 518 nm.



108

Figure 5-7: Spectral properties of a filter cube adapted to the 5-carboxyfluorescein

fluorophore (see text) [198].

Figure 5-8 represents a typical wide-field epifluorescence microscope on top of which stands

an electrochemical cell. A detailed description of the equipment has been presented in chapter

2. The illumination source is an arc lamp (Xe or Hg) and the excitation light passes through

collector lenses before it reaches the excitation filter which only allows the transmission of the

wavelengths corresponding to the fluorophore absorption band.

Figure 5-8: Schematic representation of a typical wide-field epifluorescence microscope

(courtesy of Dan Bizzotto).

The transmitted light then reaches the dichroic mirror, which is tilted at an angle of 45°

with respect to the incoming excitation light and reflects the illumination beam directly through



109

the objective and focus it onto the sample. The fluorescence generated in every direction upon

excitation is gathered by the same objective used for the excitation and transmitted through the

dichroic mirror whereas most of the scattered excitation light reaching the dichroic mirror is

reflected back to the lamp.

The fluorescence finally reaches the emission filter which only allows wavelengths

corresponding to the emission band of the fluorophore and further filter the residual excitation

light that might have gone through the dichroic mirror.

The resolution of a microscope is the shortest distance at which two separated features will

still be distinguished as distinct entities. When the light emitted from the sample goes through

the objective to form an image, it does not image a point in the object as a bright disk with

defined edges but as a slightly blurred point surrounded by diffraction rings, Airy disks as

represented on Figure 5-9. This phenomenon originates in the diffraction of light that occurs

when it goes through the aperture of the objective to reach the sample. The smaller the aperture,

the higher the effect.

Figure 5-9: Illustration of the Airy disks and the Rayleigh resolution limit [199].

It is easy to distinguished two objects when they are well separated but when the distance

between the Airy disks decreases, a limit, referred to as the Rayleigh criterion, is reached. It is

defined as the distance at which the principal maximum of the second Airy disk and the first

minimum of the first Airy disk coincide. This Rayleigh criterion giving the limit of the human

eye to see two separated points is represented on Figure 5-10.



110

Figure 5-10: Illustration of the Rayleigh criterion [200].



111

5.2 Modification of the peptide probe for fluorescence

purposes

The fluorescent dye chosen in this work is the 5-carboxyfluorescein and its structure is

shown on Figure 5-11. Derivatives of fluorescein are often used as reagents in biological

systems since they present a high absorptivity and an excellent quantum yield (Φ=0.9). The

wavelengths of excitation and emission maxima of the 5-carboxyfluorescein molecule are

respectively 494 nm and 519 nm.

Figure 5-11: Molecular structure of 5-carboxyfluorescein.

The initial thiolated p53 aptamer has been elongated by three amino acids residues, a proline,

an alanine and a lysine, to maintain the 5-carboxyfluorescein dye away from the interaction

sequence. The lateral amine group of the lysine residue was used to attach the fluorescent dye

through the formation of an amide with the carboxyl group of the dye resulting in the following

sequence H2N-CPPLSQETFSDLWKLLPAK(5-FAM)-COOH. In the rest of the work, this

sequence will be referred to as “Ap-cys-p53-fluo”.

http://www.sigmaaldrich.com/catalog/product/sigma/86826?lang=fr&region=BE



112

5.3 Interfacial behaviour of peptide monolayers under

polarisation

The interfacial behaviour of monolayers under polarisation has been studied by coupling

capacitance measurements with fluorescence microscopy. Various sets of measurements have

been performed on mixed layers formed either by one-step coadsorption or two-step adsorption,

and on a single component monolayer of peptide. Figure 5-12 presents typical curves of the

measured capacitance and fluorescence intensity upon scanning the potential from +0.1 V to -

1.25 V for mixed layers of peptide and MCB prepared by two-step adsorption (left) and

coadsorption (right). Fluorescence curves are built by plotting the mean fluorescence intensity

obtained for a full image (thus encompassing only a part of the electrode) record at a specific

potential.

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

2

4

6

8

10

12

14

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

5

10

15

20

25

30

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

2000

4000

6000

8000

10000

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.21000

1200

1400

1600

1800

2000

2200

2400

2600

(a)

(d)(c)

C/µ

F c

m-2

E vs Ag|AgCl/V

(b)

C/µ

F c

m-2

E vs Ag|AgCl/V

I flu

o/a

.u.

E vs Ag|AgCl/V

I flu

o/a

.u.

E vs Ag|AgCl/V

Figure 5-12: Representative capacitance-potential (a,b) and fluorescence-potential (c,d)

curves recorded in 10 mM phosphate buffer-pH 7.4 at peptide/MCB (1/4) mixed SAMs

obtained by coadsorption (b,d) and two-step adsorption (a,c), υ = 20 mV s-1.

On the capacity vs potential curves, two distinct behaviours are observed according to the

adsorption procedure considered. On Figure 5-12(a), respectively corresponding to a two-step

adsorption the capacity remains constant up to around -0.7 V at which a first increase in capacity

is observed and directly followed by a second increase at -1.0 V. This obvious increase in two



113

steps does not appear on Figure 5-12(b) for a coadsorbed layer. In this second case, the capacity

remains constant up to approximatively -0.7 V where a marked increase in the capacity is

noticed. In both cases, while scanning back the potential up to +0.1 V, the capacity slightly

decreases till -0.4 V but always remains higher than the initial value. This behaviour is

consistent with the reductive desorption of SAMs taking place at very negative potentials. Upon

desorption, the low permittivity layer of thiols is replaced by small polar water molecules,

which results in a higher capacitance. As the desorbed thiolated molecules diffuse away from

the electrode, only a small amount that remains in the vicinity of the surface can be readsorbed

while scanning to higher potentials, leading to a relatively higher capacity.

In the fluorescence vs potential curves, fluorescence signal remains constant from +0.1 V to -

0.5 V for both immobilisation procedures. At –0.50 V a massive increase in fluorescence is

observed up to -0.9 V for the two-step adsorption (Figure 5-12(c)) and -0.8 V for the

coadsorption (Figure 5-12(d)). It is interesting to notice that the fluorescence intensity observed

on Figure 5-12(c) is 4 times higher than that of Figure 5-12(d). Since the only fluorescent thiol

is the peptide probe, an insight on the relative amount of immobilised probe can be obtained.

This observation confirms the results obtained earlier by chronoucoulometric data, with the non

fluorescent probe, in the presence of [Ru(NH3)6]3+, indicating that a coadsorption procedure

leads to a lower density of probe molecules in the mixed layer. While sweeping back to the

initial potential, the fluorescence no longer change and reaches a constant minimum value.

It is important to notice that the fluorescence vs potential curves presented on Figure 5-12

correspond to the total fluorescence intensity of a full image recorded at a specified potential.

It has been showed that potential induced fluorescence variations of biomolecules SAMs at

metal electrodes can originate for two main reasons. Upon application of a potential, the

orientation of molecules can be modified. In the case of DNA, it has been shown that upon

application of more negative potentials, the negatively charged DNA straighten up as they are

subject to electrostatic repulsion from the negatively charged surface. On the other hand, they

are attracted to positively charged surfaces. This on and off fluorescence profile is reversible

and use the property of quenching by a metal to modulate the signal as a function of the applied

potential.

The second origin of fluorescence is related to the desorption of the adsorbed fluorescent

molecules upon application of a potential. As the fluorescently labelled molecules diffuse away

from the electrode surface, the quenching of the fluorescence by the metal is no longer effective,

resulting in a substantial increase in fluorescence. Unlike the first origin of fluorescence, this

phenomenon is not reversible since the monolayer is destroyed as the molecules diffuse away

from the electrode surface. Therefore, the increase in fluorescence is followed by a decrease

which is dependent on the diffusion rate.

As Figures 5-12(c) and (d) correspond to the mean fluorescence intensity of a full image

recorded at a specified potential, part of the information remains unexploited. Fluorescence

microscopy allows us to extract extra information. Figures 5-13 and 5-14 show a selection of

images recorded at various potentials, while scanning from +0.100 V to -1.250V and back to



114

+0.100 V, at electrodes respectively modified with Ap-cys-p53-fluo/MCB (1/4) following a

two-step adsorption and coadsorption procedure. It is important to notice that the brightness

and contrast have been adjusted with respect to the first image in order to be able to see the

progressive fluorescence variation. This explains the saturation of the fluorescence signal on

some of the images.

0.1000 V vs Ag|AgCl -0.250 V vs Ag|AgCl -0.400 V vs Ag|AgCl -0.425 V vs Ag|AgCl

-0.450 V vs Ag|AgCl -0.475 V vs Ag|AgCl -0.500 V vs Ag|AgCl -0.750 V vs Ag|AgCl


-1.125 V vs Ag|AgCl -1.175 V vs Ag|AgCl -1.175 V vs Ag|AgCl 0.100 V vs Ag|AgCl

Figure 5-13: Fluorescence images of an electrode modified by two-step adsorption with

peptide/MCB (1/4) during a reductive desorption from +0.100 V to -1.250 V. The images

(50 × objective; exposure time = 500 ms; Gain=400; Lamp intensity 30 %) are taken at

indicated potentials.



115

0.100 V vs Ag|AgCl -0.250 V vs Ag|AgCl -0.500 V vs Ag|AgCl -0.600 V vs Ag|AgCl

-0.625 V vs Ag|AgCl -0.650 V vs Ag|AgCl -0.675V vs Ag|AgCl -0.750 V vs Ag|AgCl


-1.125 V vs Ag|AgCl -1.150 V vs Ag|AgCl -0.900 V vs Ag|AgCl + 0.100 V vs Ag|AgCl

Figure 5-14: Fluorescence images of a coadsorbed peptide/MCB (1/4) modified electrode

during a reductive desorption from +0.100V to -1.250V. The images (50 × objective;

exposure time = 500 ms, Gain=400; Lamp intensity 30 %) are taken at indicated potentials.

On both Figures 5-13 and 5-14, the left image of the first row corresponding to the first

recorded image during the reductive desorption of the layer, we observe a coexistence of more

or less intense regions. This information is hidden within the fluorescence intensity curves as a



116

function of the potential since it presents a mean fluorescence profile for a full image; this value

does not convey any localised information. These variations in intensity can be interpreted

according with different hypothesis. Indeed, a higher intensity can be explained by a localised

higher density of the fluorescently labelled probe on the metal surface. This distribution can

also be explained by the presence of aggregates of the fluorescent molecule on the electrode

surface. This aggregation leading to the presence of fluorescently labelled molecules further

away from the metal surface induces a lower efficiency of the quenching by the metal.

So far, the presence of heterogeneities within self-assembled monolayers has only been studied

on DNA self-assembled monolayers. It has been shown by Murphy et al., who studied the

assembly of mixed layers composed of DNA and mercaptohexanol by in situ fluorescence

microscopy under potential control, that a two-step assembly where mercaptohexanol is

adsorbed prior to the oligonucleotides leads to more homogeneous layers than the adsorption

in two steps consisting of the immobilisation of the biomolecule followed by the passivation by

an alkanethiol [99]. AFM measurements performed by Josephs and Ye have led to the same

conclusion. Moreover, they also evidenced subpopulations of probe with various levels of probe

density [103].

In our lab, using the same technique of in situ fluorescence microscopy, we studied the

interfacial inhomogeneities of DNA mixed SAMs on gold and showed that the main factors

affecting the interfacial characteristics of mixed DNA/MCB SAMs were the substrate

crystallinity and the immobilisation procedure [104].

In this thesis, we investigate, for the first time, the interfacial characteristics of peptide SAMs

by in situ fluorescence microscopy. In Figure 5-13 presenting the images associated with the

reductive desorption for a monolayer resulting from a two-step adsorption, two distinct

increases in fluorescence are noticed. A first increase in fluorescence from -0.450 V to -0.950 V

directly followed by a second increase at -1.000V are observed. While the initial increase in

fluorescence seems to originate from the entire electrode, the second increase is more localised

and seems to arise from the regions of higher intensity. This fluorescence in two steps might be

correlated with the reductive desorption which also occurs in two well distinct steps. Both in

fluorescence and capacitance curves, we observed a first increase around -0.600 V/-0.650 V

followed by a second increase at -1.000 V although there is a small shift in the potentials at

which the increases are observed. It should be noticed that the capacitance variation arises both

from the fluorescently labelled peptide and the non fluorescent mercaptobutanol desorption

although the fluorescence signal only provides from the biomolecule.

Looking at the various images recorded in the course of the reductive desorption of the layer

immobilised by a coadsorption procedure, presented on Figure 5-14, a marked increase in

fluorescence is observed at -0.600 V until -0.950 V where it starts to decrease to reach a

minimum value where less modification of the fluorescence intensity occurs. This behaviour

can easily be correlated with the electrochemical data simultaneously recorded presented at the

beginning of this section.



117

It is also interesting to note that the zones of higher intensity observed for both films obtained

by different immobilisation procedures remains after cycling up to -1.250 V. They will be

named here as « hotspots ».

In the next sections, we propose to investigate how the fluorescence signal is influenced by the

organisation of the layer. Therefore, we will define various regions, which we will name

« regions of interest » or ROI, to study how the regions of high intensity behaves compare to

the lower ones and what are their similarities.



118

5.4 Definition of the regions of interest

To define the regions of interest, we will have to distinguish the more intense regions from

the less intense. To do so we will use the image analysis software Image J [201].

As the image obtained through the objective is more in focus than what the real image is,

a Gaussian blur filter is applied on the image. This filter allows to reduce the focalisation to a

more reliable value. It acts on each pixel of the image and applies a convolution with a Gaussian

function for smoothing. It reduces noise and redefines the edges. The more common way to

accomplish neighbourhood averaging is to replace each pixel by the average of itself and its

neighbours. This is often described as a “kernel” operation, given that its implementation can

be generalized as the sum of the pixel values in the region multiplied by a set of integer weights.

For a Gaussian blur, the set of weights approximates the profile of a Gaussian function along

any row, column or diagonal through the centre [202].

During the reductive desorption, a succession of image is recorded as various successive fixed

potentials are applied. As regions of varying intensities can be present, we want to make sure

that it consists of actual variations and not of artefacts. Therefore, the first step is to extract the

“minimum image” of a complete stack which corresponds to the minimum of each pixels of the

whole stack of images. This procedure will allow us to remove artefacts and identify

heterogeneities as real features.

Once the image is blurred and the minimum image is defined, the brightness and contrast are

adjusted. Figure 5-15 shows the minimum image of a two-step adsorbed monolayer of Ap-cys-

p53-fluo/MCB (1/4) without (a) and with (b) the application of a Gaussian blur filter.

(a) (b)

Figure 5-15: Fluorescence image without (a) and with application of a Gaussian blur filter on

an electrode modified by a two-step adsorption with Ap-cys-p53-fluo/MCB (1/4).



119

As the areas of higher intensity do not always present sharp outlines, they might appear as a

unique bigger spot although it is composed of a collection of smaller regions. To emphasise the

contours of these concentrated features, another filter can be applied to the image, the unsharp

mask. This filter subtracts a blurred copy of the image from the original to suppress large scale

variations to better show local details as illustrated on Figure 5-16.

(a) (b)

Figure 5-16: Fluorescence image before (a) and after application of an unsharp mask filter on

the system presented on Figure 5-15.

The image is now ready for the determination of the regions of interest. In this particular

case, we will have to define two types of regions of interests, meaning the hot spots regions and

the non-hot spots regions. Indeed, as we would like to investigate how the fluorescence signal

is distributed between the more and less intense areas, we will have to define a threshold value

to define the fluorescence intensity at which a pixel is consider as being part of a hotspot. To

do so a threshold value has to be adjusted. This requires to specify a range of brightness of

colour values that discriminate a feature from its surroundings, or using a region-growing

method in which selecting one point in the feature allows the software to include all touching

similar pixels. In this work, we decided to use a maximum entropy threshold. This algorithm,

based on the entropy of the histogram maximise the inter-class entropy. This technique is very

similar to the one of Otsu which assumes that the image contains two classes of pixels following

a bi-modal histogram and then calculates the optimum threshold separating the two classes so

that their inter-class variance is maximal [203]. The entropy of the brightness pattern is

calculated as the number of ways that the pattern could have been formed by rearrangement.

Figure 5-17 shows the histograms of the whole minimum image and of the selection of the

pixels associated with the hotspot regions via the maximum entropy threshold.



120

1000 2000 3000 4000 5000 6000

0

5000

10000

15000

20000

25000

30000

2500 3000 3500 4000 4500 5000

0

2

4

6

8

Counts

Pixel value

Hotspots

Counts

Pixel value

Whole image

Hotspots

Figure 5-17: Histograms of the whole minimum image and of the selection of the pixels

associated with the hotspot regions via the maximum entropy threshold.

This thresholding will allow us to create two masks, one keeping only the hotspots region and

the other keeping the rest of the image. This will allow to define how the fluorescence signal is

influenced by the presence of these higher intensity regions and discriminate this contribution

from the total fluorescence profile corresponding to the whole image presented earlier on Figure

5-12. Figure 5-18 presents a mask allowing the measurement of the fluorescence corresponding

to the hotspot regions only. The complementary mask is obtained by taking the inverse of the

selection.

Figure 5-18: Creation of a mask corresponding to the hotspots regions by setting a maximum

entropy threshold.

Once both masks are created, we can select various regions of interest within the defined hotspot

and non hotspot regions and compare their respective behaviours.



121

5.5 Reductive desorption behaviour of mixed layers

We have seen on Figure 5-12 that the reductive desorption of mixed layers of peptide and

MCB occurs in two well separated steps in the case of a two-step adsorption and in an

apparently single step in the case of a one-step coadsorption. These observations have been

confirmed by the fluorescence images recorded under polarisation and presented on Figures 5-

13 and 5-14. We have also seen that brighter areas and darker ones coexist at the surface. As a

threshold value allowing the definition of the hotspot areas has been settled, it is now possible

to investigate more deeply how these aggregates contribute to the capacitance and concomitant

fluorescence.

For each condition of immobilisation, four consecutive reductive desorption runs were

performed. During the two first reductive desorptions, the potential was scanned from +0.100

V to -1.250 V. The two subsequent cycles were recorded while scanning to more negative

potentials, respectively -1.350 V and -1.450 V. The successive curves of the capacitance as a

function of the applied potential will be interpreted concomitantly with the fluorescence

behaviour. In order to differentiate the fluorescence behaviour of the hotspots from that of the

rest of the layer, different regions of interest will be selected.

5.5.1 Case of an electrode modified by a two-step adsorption

procedure

Figure 5-19 shows four regions of interest, two of them corresponding to hotspot areas

(HS) and two others to non-hotspot areas (NHS), that have been selected and the minimum

image associated with each of the four consecutive reductive desorptions. It should be

emphasized here that, although one set of data is presented, a similar behaviour has been

observed for different experiments on multiple electrodes and for various peptide/MCB

proportions indicating that although each set has singularities, the present analysis is well

representative.



122

1st reductive

desorption

2nd reductive

desorption

3rd reductive

desorption

4th reductive

desorption

Figure 5-19: ROIs selection and minimum images corresponding to four consecutive

reductive desorptions of a peptide/MCB (1/4) mixed SAM obtained by two-step adsorption.

Figure 5-20 shows the capacitance vs potential curves of the whole electrode (right) and

the fluorescence profiles (left), extracted from the four regions of interest selected, as a function

of the applied potential with respect to the four consecutive reductive desorptions.

A first look at Figure 5-20(a) reveals that, during the first reductive desorption, a major increase

in fluorescence of similar intensity in the four ROIs, starts more negatively than -0.500 V and

a high fluorescence peak is observed at – 0.725 V while the fluorescence decreases at more

negative potentials. However, the background fluorescence is slightly higher in the two HS

regions. Looking at the corresponding capacitance curve, we notice that the signal increases

smoothly from -0.400 V until -0.700 V where a pronounced modification of the signal is

recorded.

At -1.125 V, the fluorescence profile of the HS and NHS regions varies. A small increase in

fluorescence in the HS regions is observed in contrast to the NHS regions. At about the same

potential, the capacitance curve also shows a second variation of the signal.

Looking at Figure 5-20(b), showing the data associated with a second reductive desorption, it

appears immediately that the fluorescence signal is lower of about a factor 100 indicating that

a large amount of peptide has already been removed from the surface during the first desorption

step. This is supported by the higher capacitance recorded at the beginning of the second

reductive desorption cycle at positive potentials.

Although the intensity of the first increase is lowered by about a factor of 100, the fluorescence

corresponding to the second increase remains approximatively constant. The same behaviour is

observed during the third and fourth desorption runs although the potential is swept to more

negative value, respectively -1.350 V and -1.450 V.

From the capacitance curves, we do observe that between the first and second desorption, a

modification of the layer occurs as evidenced by the lower capacitance value between the end

of the first reductive desorption and the beginning of the second reductive desorption.

Furthermore, it appears, from the increasing initial capacitance values and the persistent two

successive fluorescence increases recorded during the four consecutive desorption runs that, the

hotspots may act as a source of peptide after the first desorption cycle.



123

(a)

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

5000

10000

15000

20000

25000

30000

35000M

ean

Flu

ore

scen

ce I

nte

nsi

ty /

a.u

.

E vs Ag|AgCl / V

HS 1

HS 2

NHS 1

NHS 2

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

2

4

6

8

10

12

14

C /

µF

cm

-2

E vs Ag|AgCl / V (b)

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

400

800

1200

1600

2000

2400

2800

3200

3600

4000

Mea

n F

luore

scen

ce I

nte

nsi

ty /

a. u.

E vs Ag|AgCl / V

HS 1

HS 2

NHS 1

NHS 2

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

2

4

6

8

10

12

14

C /

µF

cm

-2

E vs Ag|AgCl / V

(c)

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

400

800

1200

1600

2000

2400

2800

3200

3600

4000

Mea

n F

luore

scen

ce I

nte

nsi

ty /

a.u

.

E vs Ag|AgCl / V

HS 1

HS 2

NHS 1

NHS 2

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

2

4

6

8

10

12

14

16

C /

µF

cm

-2

E vs Ag|AgCl / V (d)

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

400

800

1200

1600

2000

2400

2800

3200

3600

4000

4400

Mea

n F

luore

scen

ce I

nte

nsi

ty /

a.u

.

E vs Ag|AgCl / V

HS 1

HS 2

NHS 1

NHS 2

-1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

2

4

6

8

10

12

14

16

18

C /

µF

cm

-2

E vs Ag|AgCl / V

Figure 5-20 : Representative fluorescence-potential (left) and capacitance-potential (right) curves

of hotspot and non-hotspot regions associated with four consecutive reductive desorption up to -

1.250 V (a and b); -1.350 V (c); -1.450 V (d) recorded in 10 mM phosphate buffer-pH 7.4 at

peptide/MCB (1/4) mixed SAMs obtained by two-step adsorption, υ=20 mV s-1.



124

It is very impressive to note that even though the potential is scanned to very negative values,

some of the fluorescence corresponding to the first increase at -0.500 V remains and that no

decrease in the second fluorescence signal at -1.000 V associated with the hotspots regions is

observed. To better outline this distinctive behaviour between the HS and NHS regions, the

fluorescence profiles associated with to the four reductive desorptions associated with regions

HS2 and NHS2 have been superimposed on Figure 5-21.

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

5000

10000

15000

20000

25000

30000

Mea

n F

luore

scen

ce I

nte

nsi

ty /

a.u

.

E vs Ag|AgCl / V

Scan 1

Scan 2

Scan 3

Scan 4

(a)

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

5000

25000

30000

Mea

n F

luore

scen

ce I

nte

nsi

ty /

a.u

.

E vs Ag|AgCl / V

Scan 1

Scan 2

Scan 3

Scan 4

(b)

Figure 5-21: Overlay of the fluorescence vs potential curves associated with the four

consecutive reductive desorption in HS2 (a) and NHS2 (b), υ = 20 mV s-1.



125

From the data presented above, it seems to indicate that the first increase in fluorescence can

be associated with the more homogenous areas of the layer whereas the second fluorescence

increase originates from the hotspots.

5.5.2 Case of an electrode modified by a one-step coadsorption

procedure

In order to compare how the procedure of immobilisation influence the organisation of the

layer, we will now compare the fluorescence and capacity results of layers obtained by a two-

step adsorption and a one-step adsorption. Indeed, as it has been presented earlier,

electrochemical characterisation performed on layers prepared by these two procedures shows

significant differences in particular in terms of the probe density. To do so, we selected two

regions of interest being within a hotspot (HS) and in a less intense area (NHS). This selection

and the evolution of the minimum images obtained for each reductive desorption are presented

on Figure 5-22.

1st reductive

desorption

2nd reductive

desorption

3rd reductive

desorption

4th reductive

desorption

Figure 5-22: ROIs selection and minimum images corresponding to four consecutive

reductive desorptions of a peptide/MCB (1/4) mixed SAM obtained by coadsorption.

Figure 5-23 presents a typical behaviour of a mixed layer of fluorescently labelled peptide and

MCB formed by coadsorption procedure.

A first observation reveals that for the first reductive desorption, the fluorescence signal is the

10 times smaller than with a layer formed by the two-step procedure. Although fluorescence

cannot be directly associated with the concentration of species, it is consistent with the

chronocoulometric data indicating that coadsorption generates less labelled probe immobilised

at the surface than the two-step procedure. Secondly, it is interesting to note that, again, two

increases in fluorescence are observed in the hotspot region whereas only one increase is

observed in the non-hotspot area. Here again, we observed the persistence only of the hotspots

after four consecutive desorptions up to very negative potentials.



126

Another different behaviour of the film formed by coadsorption is that no significant

modification of the fluorescence signal is observable after the first reductive desorption

indicating that a large majority of the peptide probe is removed and diffuses to the bulk. This is

also confirmed by the absence of significant modifications of the capacitance value respectively

to the three next successive desorptions indicating that a minimum value has been reached.

Looking at the fourth reductive desorption, we observe however a small increase in

fluorescence in the NHS region at the same potential as that observed in the HS region. We

assume that this signal is due to the apparition of bubbles at the electrode surface due to the

reaction of hydrogen evolution, as we are scanning to very low potentials, rather than to a

significant contribution.



127

(a)

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

500

1000

1500

2000

2500

3000

3500

Mea

n F

luo

resc

ence

In

ten

sity

/ a

.u.

E vs Ag|AgCl / V

HS

NHS

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

5

10

15

20

25

30

C /

µF

cm

-2

E vs Ag|AgCl / V

(b)

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

500

1000

1500

2000

Mea

n F

luo

resc

ence

In

ten

sity

/ a

.u.

E vs Ag|AgCl / V

HS

NHS

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

5

10

15

20

25

30

35

40

C /

µF

cm

-2

E vs Ag|AgCl / V

(c)

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

500

1000

1500

2000

Mea

n F

luo

resc

ence

In

ten

sity

/ a

.u.

E vs Ag|AgCl / V

HS

NHS

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

5

10

15

20

25

30

35

40

C /

µF

cm

-2

E vs Ag|AgCl / V

(d )

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

500

1000

1500

2000

Mea

n F

luo

resc

ence

In

ten

sity

/ a

.u.

E vs Ag|AgCl / V

HS

NHS

-1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

5

10

15

20

25

30

35

40

45

C /

µF

cm

-2

E vs Ag|AgCl / V

Figure 5-23: Representative fluorescence-potential (left) and capacitance-potential (right) curves

of hotspot and non-hotspot regions associated with four consecutive reductive desorption up to -

1.250 V (a and b); -1.350 V (c); -1.450 V (d) recorded in 10 mM phosphate buffer-pH 7.4 at

peptide/MCB (1/4) mixed SAMs obtained by one-step coadsorption, υ= 20 mV s-1.



128

5.6 Interfacial behaviour of a single component layer

composed of peptide

As the presence of distinct regions observed in the fluorescence imaging of layers formed

by two different procedures could arise from a segregation of phases between the

mercaptobutanol diluent and the peptide aptamer probe, we decided to examine the

fluorescence signal in the absence of diluent. The same strategy was used, namely the

succession of two reductive desorptions from +0.100 V to -1.250V followed by two consecutive

desorptions up to -1.350 V and -1.450 V but, this time, the gold electrode was exclusively

modified by labelled thiolated peptide. Figure 5-24 presents an image of a gold electrode

modified by the peptide layer. As for mixed layers, a coexistence of more and less intense

regions is observed indicating that the presence of hotspots cannot simply be explained by a

phase segregation due to the presence of MCB in the mixed monolayers.

Figure 5-24: Fluorescence image acquired at open circuit potential (50 × objective, exposure

time = 500 ms) for polycrystalline gold surface modified by a SAM of peptide.

Figure 5-25 presents a typical curve of the total fluorescence as a function of the applied

potential for a full image of a self-assembled monolayer exclusively composed of the

fluorescently labelled peptide. Two significant increases in fluorescence are observed during

the first reductive desorption whereas only one significative increase is observed at more

negative potential for the additional successive scans.



129

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

200

400

600

800

1000

1200

1400

1600

Scan 1

Scan 2

Scan 3

Scan 4

Mea

n F

luore

scen

ce I

nte

nsi

ty /

a.u

.

E vs Ag|AgCl / V

Figure 5- 25: Representative fluorescence-potential curves of four successive reductive

desorption runs recorded in 10 mM phosphate buffer-pH 7.4 at a peptide alone SAM modified

electrode.

A first increase is observed from -0.2 V until -0.7 V where the fluorescence starts to

decrease up to -1.0 V before a second increase is observed around -1.0 V. A similar two-step

fluorescence behaviour was observed in the case of mixed layers although the increase started

at around -0.5 V. The onset of the fluorescence at a more positive potential could be explained

by the absence of mercaptobutanol. One of the role of this diluting molecule is to prevent from

the nonspecific adsorption of the biomolecules on gold. Therefore, in its absence, non-

specifically adsorbed molecules might be present at the electrode surface, the lower stability of

this anchoring leading to an earlier desorption of those species upon the application of a

negative potential.

Another hypothesis could be the second role of MCB which is to stabilise the layer by diluting

the negatively charged peptide aptamer to decrease the electrostatic repulsion between the

strands. The evolution of the capacitance as a function of the applied potential presented on

Figure 5-26 supports this assumption since that capacitance already starts to significantly

increase at -0.4 V compared to -0.6 V and -0.7 V respectively for a mixed layers.



130

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.20

2

4

6

8

10

12

14

16

18

C /

µF

cm

-2

E vs Ag|AgCl / V

Figure 5-26: Representative capacity-potential curves of the first reductive desorption from

+0.100 V to -1.250 V recorded in 10 mM phosphate buffer-pH 7.4 at a peptide SAM modified

electrode.

It is interesting to note that the capacitance also rises in two successive steps. On the forward

scan, a first increase is observed between -0.4 V and -0.9 V where the curvature changes and a

second increase is observed until the potential is scanned backwards.

More information will be obtained by selecting two regions of interest in a hotspot region and

a non-hotspot region. The selection and associated results are presented on Figure 5-27 and 5-

28, respectively.

Figure 5-27: ROIs selection and minimum image corresponding to the reductive desorption of

a peptide SAM.



131

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2400

600

800

1000

1200

1400

1600

1800

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2

500

600

700

800

900

1000

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2

500

600

700

800

900

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2

500

600

700

800

900(c)

(b)

Mea

n F

luore

scen

ce I

nte

nsi

ty /

a.u

.

E vs Ag|AgCl / V

HS

NHS

Mea

n F

luore

scen

ce I

nte

nsi

ty /

a.u

.

E vs Ag|AgCl / V

HS

NHS

(d)

Mea

n F

luore

scen

ce I

nte

nsi

ty /

a.u

.

E vs Ag|AgCl / V

HS

NHS

Mea

n F

luore

scen

ce I

nte

nsi

ty /

a.u

.

E vs Ag|AgCl / V

HS

NHS

(a)

Figure 5-28: Representative fluorescence-potential curves associated with the hotspot and

non-hotspot areas for four successive reductive desorptions from +0.100 V to -1.250 V (a,b), -

1.350 V (c) and -1.450 V (d) recorded in 10 mM phosphate buffer-pH 7.4 at a peptide SAM

modified electrode.

From the fluorescence vs potential curves shown on Figure 5-28(a), two consecutive

increases are observed. Contrary to the case of the mixed films formed by a two-step adsorption,

the first increase has almost disappeared after the first reductive desorption, which indicates

that the molecules have clearly diffused from the electrode surface to the bulk.

Another interesting feature to underline that differs from the behaviour of mixed layers is the

presence of a second peak, both in the hotspot and non-hotspot regions irrespective of the

number of desorption cycles. After the first desorption cycle, the second increase solely remains

allowing a better assignment of the potential where it appears. From the curves associated to

the last three desorptions, the fluorescence increase appears at -0.8 V in the hotspot region and

at about -1.1 V in the non-hotspot region. From Figure 5-29, it seems that the fluorescence

associated with the non-hotspot region is mainly related to the diffusion in every directions of

the fluorescence initiated by the hotspots.



132




Figure 5-29: Fluorescence images of a gold electrode modified by the peptide alone for the

second reductive desorption. The images (50 × objective; exposure time = 500 ms) are taken

at indicated potentials.

Note, once again, the persistence of the hotspot regions after four successive cycles of reductive

desorption to very negative potentials.

Further work is required to study these hotspots and we propose to investigate what could

influence the presence of these regions.



133

5.7 Study of the heterogeneities of the SAMs

As clearly evidence by the images, the fluorescence is not uniformly distributed on the

electrode and a coexistence of more or less intense regions are observed prior to the reductive

desorption. In the case of DNA SAMs, Murphy et al. suggested that these brighter regions could

be related to higher density of the fluorescent molecules or to their aggregation resulting in the

presence of labelled molecules further from the surface [99]. A first hypothesis regarding the

origin of this inhomogeneous distribution could be the structure of the electrode such as the

presence of defects. To explore this possibility, a bright field image has been taken prior to

every experiment to be able to compare the structure of the electrode to the distribution of the

hot spots. Figure 5-30 presents the images of a same area recorded in bright field and

fluorescence mode of a Ap-cys-p53-fluo/MCB (1/4) modified electrode prepared by a two-step

adsorption procedure.

(a) (b)

Figure 5-30: (a) Bright field (10 × objective, exposure time = 100 ms, Lamp Intensity= 10 %,

Gain = 400) and (b) fluorescence (10 × objective, exposure time = 500 ms, Lamp Intensity

=30 %, Gain =400) acquired at open circuit potential for a mixed SAM of Ap-cys-p53-

fluo/MCB (1/4) prepared by a two-step adsorption procedure.

On Figure 5-30(b) we first selected two regions of interest, ROI 1 and ROI 2, corresponding

to areas of high fluorescence intensity. Upon reporting those regions on the bright field image,

no major defect can be associated with the presence of these brighter fluorescence features.

Moreover, the selection of a third region, ROI 3, on Figure 5-30(a), presenting a succession of

steps and terrace, does not show any brighter spot when the selection is reported on the

fluorescence image. This indicates that the presence of brighter fluorescence regions in the self-

assembled monolayer cannot be directly correlated to the presence of defects at the substrate.



134

This comparison has been realised on a variety of immobilisation conditions on several

electrodes, leading always to this same conclusion.

It was also suspected that these hotspots could be correlated to the substrate crystallinity

but experiments carried out, in our lab, with SAMs of fluorescently labelled oligonucleotides

on polycrystalline, single crystal (111) and single crystal (210) gold electrodes have shown no

influence on the occurrence of these features as hotspots were systematically present [104].

Fluorescence measurements carried out with a single crystal (210) modified in two steps by a

peptide/MCB (2/3) layer have confirmed these results as presented on Figure 5-31. AFM

measurements performed on Arrandee (111) gold plates modified by a peptide layer have also

confirmed the same statement. AFM measurements will be discussed in more details in the

future developments of this work.

Figure 5-31: Fluorescence image (10 × objective, exposure time = 2 s) acquired at open

circuit potential for a two-step adsorbed Ap-cys-p53-fluo/MCB (2/3) SAM on a gold single

crystal (210).

As Murphy suggested that the presence of hotspots could be related to the formation of

aggregates of labelled biomolecules on the gold substrate and, as urea is often used for its

denaturing effect on protein, we tried to evaluate whether it could have an influence on the

hotspots. To do so we prepared a peptide SAM and proceeded to a reductive desorption from

+0.1 V to -1.250V in order to only keep the hotspots on the gold surface. We, then, exposed the

electrode surface to a denaturing solution of 8 M urea overnight. As negative control, we

performed the same experiment, but instead of urea, the electrode was exposed to a 1 M

phosphate buffer - pH 7.4. Figure 5-32 shows an OCP image of the electrode surface before

and after an overnight contact with 1 M phosphate buffer- pH 7.4 (a, b) and with 8 M urea (c,d).



135

(a) (b)

(c) (d)

Figure 5-32: Fluorescence images (10 x objective, exposure time = 1 s) acquired at open

circuit potential for a peptide SAM before and after overnight contact with 1 M phosphate

buffer – pH 7.4 (a,b) and 8 M urea (c,d).

It can be seen on Figure 5-32(d) that the aggregates observed on Figure 5-32(c) remain after

being treated overnight with urea. No significant modification is observed in comparison with

the effect of phosphate buffer shown on Figure 5-32(a, b). The effect of ultrasounds has also

been examined by exposing the electrode for 10 minutes in an ultrasounds bath but no

noticeable effect has been detected.

We also tried to immobilise a fluorescently labelled non thiolated probe, corresponding to

the p53 peptide labelled probe without additional cysteine residue in N-terminal position, to see

if hotspots would be present. Figure 5-33 shows 10× magnification fluorescence images on this

probe immobilised overnight in 1 M phosphate buffer-pH 7.4 before and after performing a

potential sweep experiment from +0.100 V to –1.250 V in 10 mM phosphate buffer pH 7.4.



136

(a) (b)

Figure 5-33: Fluorescence images (10 × objective, exposure time = 500 ms) acquired at open

circuit potential for an unthiolated peptide SAM before (a) and after (b) reductive desorption

from +0.100 V to -1.250 V.

Although less hotspots are observed than for a thiolated probe, significant aggregates are

observed and these remain through desorption. This indicates that these aggregates are not only

due to thiol-gold bonding but to other interactions between the peptide probes such as hydrogen

bonding or hydrophobic effects keeping them in the vicinity of the surface and preventing them

from diffusing away from the electrode surface upon the application of a potential.



137

5.8 AFM characterisation of the peptide SAMs

AFM allows to determine the nanoscale distribution of the thiolated molecules immobilised

at the electrode surface and is the only technique that reaches a sufficient lateral resolution to

localise individual molecules. These abilities makes it one of the most prized technique in the

study of the organisation of self-assembled monolayers. Different parameters can be obtained

as the roughness. Since AFM gives information on the relative height at which the molecules

are distributed on the surface and that the hotspots observed in fluorescence are assumed to be

aggregates which relative height from the electrode surface should be higher, we performed

non-contact mode AFM measurements to evaluate whether the aggregates observed in

fluorescence microscopy could be correlated to features observed via this highly resolved

technique. Figure 5-34(a) presents a 50 × magnification fluorescence image of a peptide/MCB

(2/3) electrode modified by a two-step adsorption. Using the corresponding bright field image

of the electrode, we were able to position the cantilever in the area in the upper right corner of

the fluorescence image, to perform an AFM imaging, at air, of this region. The image recorded

is shown on Figure 5-34(b).

(a)

(b)

Figure 5-34: Fluorescence image (50 × objective, exposure time = 1 s) acquired at open

circuit potential for a two-step adsorbed Ap-cys-p53-fluo/MCB (2/3) SAM on a gold

electrode (a) and AFM image of this same region (b).

By comparing the distance and angles between different hotspots, we were able to correlate the

hotspots observed in fluorescence with features observed in AFM as represented by the

coloured lines on Figure 5-34. The height profiles of a few aggregates have been obtained by

plotting the height as a function of the cross section of the hotspots as shown on Figure 5-35.

Note that the hotspot at the extreme right has been set as 1, the others being numbered

clockwise.



138

0 1 2 3 4 5 6 7

-50

0

50

100

150

200

250

300

350

Hei

ght

/ nm

Section / µm

HS 1

HS 3

HS 4

Figure 5-35: Height profiles of cross-sections performed on some aggregates observed on

AFM images recorded on a Ap-cys-p53-fluo/MCB (2/3) modified gold electrode obtained by

two-step adsorption.

It can be seen that the height of the hotspots varies between roughly 150 and 300 nm. This is

interesting since we have shown on Figure 3-30 that the quenching of fluorescence is no longer

effective for a fluorescein fluorophore, at a distance of 150 nm from the electrode, allowing the

visualisation of the labelled molecules by fluorescence microscopy. These observations further

support our assumption on the origin of the hotspots as resulting from an aggregation of

fluorescent biomolecule at the electrode surface.

Besides imaging, AFM, as its name suggests, also allows the direct measurement of tip-

sample interaction forces as a function of the gap between the tip and sample. This measurement

gives information on the adhesion of the sample on the substrate and corresponds to the

minimum of the force-distance curve to finally obtain a topographic image of the adhesion as a

function of the distance. Since we have tried, via fluorescence measurements, to contribute to

the study of the organisation of self-assembled monolayers of peptide mixed or not with MCB

and since we have shown that the hotspots observed in fluorescence can be correlated to the

features observed via AFM, we decided to perform experiments on four different samples to

obtain information both on the distribution and adhesion of the layers. The prepared layers

were:

a monolayer of 4-mercaptobutan-1-ol (MCB)

a layer exclusively composed of peptide (Ap-cys-p53-fluo)

a mixed layer composed of mercaptobutanol and Ap-cys-p53-fluo (2/3) formed in one

step



139

a mixed layer composed of mercaptobutanol and Ap-cys-p53-fluo (2/3) formed in two

steps

AFM contrary to fluorescence allows the visualisation of the organisation of both fluorescent

and non fluorescent molecules. Furthermore, its higher resolution should allow a sharper

visualisation of the organisation. Therefore, we propose to compare the height vs distance and

force vs distance images obtained for the four samples as presented on Figure 5-36.

(a)

(b)



140

(c)

(d)

Figure 5-36: Height vs distance and force vs distance image obtained by atomic force

microscopy of a layer of MCB (a), Ap-cys-p53-fluo (b), Ap-cys-p53-fluo/MCB formed in one

step (c), Ap-cys-p53-fluo/MCB formed in two steps (d).

It can be seen on Figure 5-36 that the four layers present different behaviours both in terms of

height distribution and adhesion. Figure 5-36(a) corresponding to a monolayer of MCB presents

a quite homogeneous organisation. Indeed from the adhesion image, we can assume that the

higher patterns observed originates from dried drops of electrolyte rather than from the

alkanethiol molecules. The variations observed in the force distribution however indicate that

the MCB is probably assembled in a succession of well organised domains rather than in one

major domain.

Figure 5-36(b) illustrating a layer of peptide presents a highly homogeneous organisation as

can be seen from both height and adhesion images. Indeed, the amplitude of the height variation

is smaller than in any of the other three adsorbed layers and the same observation can be made



141

for the adhesion image. This indicates that the adsorption of the peptide only results in the

formation of highly compact layers which is in agreement with the electrochemical results

previously shown in this work presenting a complete inhibition of the electron transfer

associated with the oxidation and reduction processes of the [Fe(CN)6]3-/4- redox couple.

It is also very impressive to observe that the organisation of the mixed layers is quite different

from the peptide or MCB layers but also from each other. Indeed, the adhesion patterns are, in

both cases, very heterogeneous compared to the peptide or to MCB but the organisation of these

patterns also strongly varies. The force obtained for the one-step adsorbed layer cannot,

however, be compared to the others since we had to change cantilevers for this experiment.

Besides, it can be seen on each image that the higher patterns present smaller adhesion than the

rest of the sample.

At this stage of the work, it is not possible to go any further in the interpretation of these data.

However, considering the variations observed from one procedure of adsorption to the other,

both via electrochemical methods, fluorescence and atomic force microscopy, it would be very

interesting to pursue this investigation. For example, a similar coupling to the one used in this

work for fluorescence measurements would allow a more accurate and more systematic study

of the organisation of the peptides layers and the origin of the strong adsorption of the

aggregates.

142

Chapter 6 Conclusions

143

Chapter 6: Conclusions

6.1 General conclusions

In the first part of this thesis, we have shown that p53-based peptide aptamer sequences

can be assembled on gold through addition of a cysteine residue to allow its covalent anchoring

via the thiol side chain of the amino acid. The immobilisation of the peptide probe has been

performed according to distinct procedures in which the biomolecule was adsorbed alone or in

the presence of a diluent, the 4-mercaptobutan-1-ol (MCB).

Experiments performed in the presence of the redox complex [Ru(NH3)6]3+, electrostatically

interacting with the negatively charged peptide, have evidenced that the simultaneous

adsorption of these two thiolated molecules leads to a lower peptide ratio in the monolayer

(about 10 %) compared to the two-step adsorption in which the peptide is absorbed prior to the

MCB (about 15 %). The adsorption of the peptide alone leads to the most concentrated layers

with twice the amount of biomolecule compared to the two-step adsorption. Furthermore, we

showed using electrochemical impedance spectroscopy in the presence of the redox couple

[Fe(CN)6]3-/4- that the adsorption of a layer exclusively composed of the peptide leads to a

complete inhibition of the electron transfer. This inhibition, not observed for mixed layers, has

been related to the presence of the negatively charged peptide aptamer. Indeed, in mixed layers,

channels are preserved through the film by the mercaptobutanol and the electron transfer

kinetics is merely affected. The negatively charged peptide layer forms thus a barrier to the

electronic transfer, and totally inhibits the signal. AFM measurements have confirmed the

compact organisation of the layers exclusively composed of peptide. The dramatically different

behaviours towards electron transfer observed between the mixed layers and the layer of peptide

underlines the attention that should be given to the influence of the immobilisation procedure

on the organisation of the resulting self-assembled monolayers.

In the second part of this work, a new strategy of detection of the protein MDM2, relying

on the immobilisation of a peptide aptamer based on the interaction sequence of the p53 protein

with the MDM2 protein has been implemented. This method relies on the analysis of the

variation of the charge transfer resistance (ΔRct) of the [Fe(CN)6]3-/4- redox couple at the

modified interface by electrochemical impedance spectroscopy. Whereas the three adsorption

procedures have been tested, the monolayers composed of the sole peptide probe have shown

the most promising results. Indeed, the inhibition of the electronic transfer, evidenced in

absence of diluent, was used to follow the interaction between the peptide probe and MDM2.

The study of the behaviour of MDM2 at gold electrodes via cyclic voltammetry,


144

electrochemical impedance spectroscopy and in situ fluorescence microscopy have shown that

the modification of the charge transfer resistance upon contact with the target protein is not

resulting from the desorption of the peptide probe. Quartz crystal microbalance measurements

performed on gold-coated quartz crystals modified by a peptide layer have confirmed the

interaction between MDM2 and the probe. Negative controls performed with bovine serum

albumin, fibrinogen and cytochrome c further supports the strategy. At the highest protein

surface coverage, a ratio of 1 MDM2 for 10 Ap-cys-p53 probes has been obtained.

The analytical performance of the detection method has been assessed. Experiments have been

performed by exposing electrodes modified by the peptide to various concentration of MDM2

and the limit of detection (LOD) and limit of quantification (LOQ) have been determined based

on the same negative controls as those used in QCM. A limit of detection of 12 pM,

corresponding to 0.69 ng mL-1 and a limit of quantification of 19 pM, corresponding to

1.08 ng mL-1 have been determined. The detection range of 1.08 to 58 ng mL-1 and LOD of

0.69 ng mL-1 obtained with the transducer elaborated in this work indicates that, the

immobilisation of a peptide aptamer combined with the impedimetric detection can compete

with the performance of ELISA kits. Therefore, the strategy developed in this work, relying on

the use of a peptide sequence whose selection is based on the interaction sequence of a protein

with the considered target seems very promising.

In the third part of this work, we studied, via in situ fluorescence microscopy, the influence

of the adsorption procedure on the organisation of the peptide monolayers. For fluorescence

purposes, the initial probe has been labelled with a 5-carboxyfluorescein dye. In each

immobilisation procedure, a coexistence of more or less intense fluorescent regions was

observed. The brighter areas were referred to as “hotspots”. Four successive reductive

desorptions were performed to study the behaviour of the monolayers.

In mixed layers, two increases in the fluorescence signal at about -0.6 V and -1.0 V were

recorded during the first desorption cycle. However, the fluorescence intensity observed for a

layer obtained by a two-step adsorption was about 10 times higher compared to those formed

by coadsorption. This observation indicates a higher peptide concentration as evidenced by our

electrochemical results in the presence of [Ru(NH3)6]3+. From the second cycle, variations were

observed in the behaviour of mixed layers. Indeed, whereas the signal intensity associated with

the first fluorescence increase diminishes by a factor 10 in the case of a two-step adsorption, it

completely vanishes in coadsorption. However it is interesting to notice that the fluorescence

intensity of the second increase remains, in both cases, approximatively constant even after four

reductive desorption cycles. In addition, the hotspots also remain after four cycles of reductive

desorption.

The definition of regions of interest allowed us to evidence different fluorescence behaviours

for the hotspot (HS) and non hotspot (NHS) regions. We were able to assign the first increase

in fluorescence to the less intense areas of the layer and the second fluorescence increase to the

hotspots.


145

The presence of hotspots in the layers exclusively composed of the peptide probe indicates that

the heterogeneities are not related to a phase segregation between the biomolecule and the

MCB. As in mixed layers, two increases in the fluorescence signal were observed although the

desorption started at a more positive potential (-0.2 V) witnessing the stabilising effect of the

MCB in mixed layers. As for the hotspots, the fluorescence intensity associated with the second

increase remained after four consecutive desorption cycles.

As Murphy et al. [99] suggested that these brighter regions could be related to higher density

of the fluorescent molecules or to their aggregation resulting in the presence of labelled

molecules further from the surface, different hypotheses regarding their origin were tested.

However, no influence of the presence of defects or of the crystallinity of the substrate were

evidenced on the presence of aggregates in the peptide monolayers. Similarly, no effect of a

denaturing solution of urea or of the use of ultrasounds were observed on the presence of

hotspots. However, atomic force microscopy measurements (AFM) allowed us to correlate the

brighter regions observed in fluorescence with higher features imaged in AFM. This supported

the hypothesis that the hotspots actually are aggregates of peptide on the surface.

6.2 Prospects

Beyond the results presented in this work, various prospects are worth to be considered.

Indeed, we have shown that a sensing strategy relying on the immobilisation on gold of a

peptide probe based on the natural sequence of interaction of the protein p53 with the protein

MDM2, can be used to follow their interaction. Therefore, it would be interesting, in future

work, to further assess the performance of this probe towards its target using other transducers.

For instance, the immobilisation of this same peptide on gold could be used to detect the protein

MDM2 via surface plasmon resonance (SPR) or quartz crystal microbalance (QCM). Besides,

the use of gold nanoparticles as support could also be considered.

Secondly, new peptide/protein couples should be tested to evaluate whether other

interaction sequences might be suitable for protein detection and further support the strategy

implemented in this thesis.

Furthermore, infrared spectroscopy might elucidate whether the complete inhibition of the

electron transfer observed in the case of monolayers exclusively composed of the p53 peptide

can be related to a secondary structure of the peptide probe. Indeed, in the work developed by

Gatto [109, 110], self-assembly of helices were shown to block the electron transfer.

The influence on the electron transfer inhibition of a modification of the p53 aptamer

sequence by a variety of spacer inserted between the cysteine anchor and the probe sequence

would also be worth investigation. In the same idea, the length of the interaction sequence or

the addition of amino acids as the α-aminoisobutyric acid residue (Aib) known to force the

sequence to take a rigid 310 helix structure could be modified. These modifications would also


146

help to understand the organisation of the monolayer in relation to the sequence of the probe

and/or its structure.

The deeper understanding of the organisation of the monolayer might also be obtained by

further atomic force microscopy measurements. Coupling AFM measurements in solution with

electrochemistry can provide further insights into the correlation between the interfacial

properties of the peptide layers and the electron transfer. Besides complementary measurements

are necessary to understand the origin of the aggregates and whether the substrate is a driving

force for the formation of aggregates in the monolayers.

147

References

[1] G. Evtugyn, Biosensors: Essentials, Lecture Notes in Chemistry ed., vol. 84, Berlin Heidelberg:

Springer-Verlag, 2014.

[2] R. Vargas-Bernal, E. Rodriguez-Miranda and G. Herrera-Pérez, “Chapter 14: Evolution and

Expectations of Enzymatic Biosensors for Pesticides,” in Pesticides - Advances in Chemical and

Botanical Pesticides, R. P. Soundararajan, Ed., InTech, 2012, pp. 329-356.

[3] M. Zourob, Recognition Receptors in Biosensors, New York: Springer, 2010.

[4] M. A. Cooper, Label-free biosensors: techniques and applications, New York: Cambridge

University Press, 2009.

[5] M. B. Gu and H.-S. Kim, Biosensors based on Aptamers and Enzymes, Advances in Biochemical

Engineering/Biotechnology ed., vol. 140, T. Scheper, Ed., Berlin Heidelberg: Springer, 2014.

[6] J. Janata, Principles of chemical sensors, 2nd ed., Boston: Springer, 2009.

[7] K. R. Rogers and A. Mulchandani, “Principles of Affinity-Based Biosensors,” in Affinity

Biosensors: Techniques and Protocols, Methods in Biotechnology ed., vol. 7, Totowa, Humana

Press, 1998, pp. 3-18.

[8] E. Palecek, J. Tkac, M. Bartosik, T. Bertok, V. Ostatna and J. Palecek, “Electrochemistry of

Nonconjugated Proteins and Glycoproteins.Toward Sensors for Biomedicine and Glycomics,”

Chemical Reviews, vol. 115, pp. 2045-2108, 2015.

[9] S. Hu , Q. Lu and Y. Xu, “Chaper 17: Biosensors based on direct electron transfer of protein,” in

Electrochemical sensors, biosensors and their medical applications, Academic Press, 2011.

[10] M. Cretich, F. Damin, G. Pirri and M. Chiari, “Protein and peptide arrays: Recent trends and new

directions,” Biomolecular Engeneering, vol. 23, pp. 77-88, 2006.

[11] D. R. Thévenot, K. Toth, R. A. Durst and G. S. Wilson, “Electrochemical Biosensors:

Recommended Definitions and Classification,” Pure Applied Chemistry, vol. 71, pp. 2333 - 2348,

1999.

[12] D. Mimica, J. H. Zagal and F. Bedioui, “Electrocatalysis of nitric oxide reduction by hemoglobin

entrapped in surfactant films,” Electochemical Communications, vol. 3, pp. 435-438, 2001.

[13] F. Lisdat, B. Ge, E. Ehrentreich-Förster, R. Reszka and F. W. Scheller, “Superoxide dismutase

activity measurement using cytochrome c-modified electrode,” Analytical Chemistry, vol. 71,

pp. 1359-1365, 1999.

[14] C. Fan, G. Li, J. Zhu and D. Zhu, “A reagentless nitric oxide biosensor based on hemoglobin-DNA

films,” Analytica Chimica Acta, vol. 423, pp. 95-100, 2000.

148

[15] U. Wollenberger, R. Spricigo, S. Leimkühler and K. Schröder, “Protein Electrodes with Direct

Electrochemical Communication,” in Biosensing for the 21st Century, vol. 109, Berlin

Heidelberg, Springer, 2008, pp. 19-64.

[16] F. Lisdat and F. W. Scheller, “Principles of Sensorial Radical Detection-A Minireview,” Analytical

Letters, vol. 33, pp. 1-16, 2000.

[17] H. Ju and D. Leech, “Electrochemical study of a metallothionein modified gold disk electrode

and its action on Hg2+ cations,” Journal of Electroanalytical Chemistry, vol. 484, pp. 150-156,

2000.

[18] F. Lisdat and I. Karube, “Copper proteins immobilised on gold electrodes for (bio)analytical

studies,” Biosensors and Bioelectronics, vol. 17, pp. 1051-1057, 2002.

[19] E. Chow and J. J. Gooding, “Peptide Modified Electrodes as Electrochemical Metal Ion Sensors,”

Electroanalysis, vol. 18, pp. 1437-1448, 2006.

[20] P. Corbisier, D. Van Der Lelie, B. Borremans, A. Provoost, V. de Lorenzo, N. L. Brown, J. R. Lloyd,

J. L. Hobman, E. Csöregi, G. Johansson and B. Mattiasson, “Whole cell- and protein-based

biosensors for the detection of bioavailable heavy metals in environmental samples,” Analytica

Chimica Acta, vol. 387, pp. 235-244, 1999.

[21] R. S. Yalow and S. A. Berson, “Assay of plasma insulin in human subject by immunological

methods,” Nature, vol. 184, pp. 1648-1649, 1959.

[22] X. Pei, B. Zhang, J. Tang, B. Liu, W. Lai and D. Tang, “Sandwich-type immunosensors and

immunoassays exploiting nanostructure labels: A review,” 2013, vol. 758, pp. 1-18, Analytica

Chimica Acta.

[23] W. Ying, Y. Su, X. Zhu, G. Liu and C. Fan, “Development of electrochemical immunosensors

towards point of care diagnostics,” Biosensors and Bioelectronics, vol. 47, pp. 1-11, 2013.

[24] G. Liu and Y. Lin, “Nanomaterial labels in electrochemical immunosensors and immunoassays,”

Talanta, vol. 74, pp. 308-317, 2007.

[25] F. Yan, J. Wu, F. Tan, Y. Yan and H. Ju, “A rapid simple method for ultrasensitive electrochemical

immunoassay of protein by an electric field-driven strategy,” Analytica Chimica Acta, vol. 644,

pp. 36-41, 2009.

[26] C.-C. Lin, L.-C. Chen, C.-H. Huang, S.-J. Ding, C.-C. Chang and H.-C. Chang, “Development of the

multi-functionalized gold nanoparticles with electrochemical-based immunoassy for protein A

detection,” Journal of Electroanalytical Chemistry, Vols. 619-620, pp. 39-45, 2008.

[27] A. Vasudev, A. Kaushik and S. Bhansali, “Electrochemical immunosensor for label free epidermal

growth factor receptor (EGFR) detection,” Biosensors and Bioelectronics, vol. 39, pp. 300-305,

2013.

[28] X. Liu, P. A. Duckworth and D. K. Wong, “Square Wave Voltammetry versus electrochemical

impedance spectroscopy as a rapid detection technique at electrochemical immunosensors,”

Biosensors and Bioelectronics, vol. 25, pp. 1467-1473, 2010.

149

[29] A. Warsinke, W. Stöcklein, E. Leupold, E. Micheel and F. W. Scheller, “Chapter 14:

Electrochemical Immunosensors on the Route to Proteomic Chips,” in Electrochemistry of

Nucleic Acids and Proteins-Towards ELectrochemical sensors for Genomics and Proteomics, E.

Palecek, F. Scheller and J. Wang, Eds., Amsterdam, Elsevier, 2005, pp. 451- 484.

[30] W. Lu, H. Zhao and G. G. Wallace, “Pulsed electrochemical detection of proteins using

conducting polymer based sensors,” Analytica Chimica Acta, vol. 315, pp. 27-32, 1995.

[31] D. Ozkan, P. Kara, K. Kerman, B. Meric, A. Erdem, F. Jelen, P. E. Nielsen and M. Ozsoz, “DNA and

PNA sensing on mercury and carbon electrodes by using methylene blue as an electrochemical

label,” Bioelectrochemistry, vol. 58, pp. 119-126, 2002.

[32] J. Wang, X. Cai, G. Rivas, H. Shiraishi, P. A. M. Farias and N. Donta, “DNA Electrochemical

Biosensor for the Detection of Short DNA Sequences Related to the Human Immunodeficiency

Virus,” Analytical Chemistry, vol. 68, pp. 2629-2634, 1996.

[33] B. Piro, J. Haccoun, M. Pham, L. Tran, A. Rubin, H. Perrot and C. Gabrielli, “Study of the DNA

hybridization transduction behavior of a quinone-containing electroactive polymer by cyclic

voltametry and electrochemical impedance spectroscopy,” Journal of Electroanalytical

Chemistry, vol. 577, pp. 155-165, 2005.

[34] A. M. V. Mohan, K. K. Aswini, A. M. Starvin and V. M. Biju, “Amperometric detection of glucose

using Prussian blue-graphene oxide modified platinum electrode,” Analytical Methods, vol. 5,

pp. 1764-1770, 2013.

[35] C. G. Begley and L. M. Ellis, “Raise standards for preclinical cancer research,” Nature, vol. 483,

pp. 531-533, 2012.

[36] I. Prassas and E. P. Diamandis, “Translational researchers beware! Unreliable commercial

immunoassays (ELISAs) can jeopardize your research,” Clinical Chemistry and Laboratory

Medicine, vol. 52, p. 765–766, 2014.

[37] M. Baker, “Blame it on the antibodies,” Nature, vol. 521, pp. 274-276, 2015.

[38] S. Puertas, M. Moros, R. Fernandez-Pacheco, M. R. Ibarra, V. Grazu and J. de la Fuente,

“Designing novel nano-immunoassays: antibody orientation versus sensitivity,” Journal of

Physics D: Applied Physics, vol. 43, p. 474012 (8pp), 2010.

[39] B. Lu, M. R. Smyth and R. O'Kennedy, “Oriented immobilization of antibodies and its

applications in immunoassays and immunosensors,” Analyst, vol. 121, pp. 29R-32R, 1996.

[40] F. Hoppe-Seyler, I. Cmkovic-Mertens, E. Tomai and K. Butz, “Peptide Aptamers: Specific

Inhibitors of Protien Function,” Current Molecular Medicine, vol. 4, pp. 539-538, 2004.

[41] I. C. Baines and P. Colas, “Peptide aptamers as guides for small-molecule drug discovery,” Drug

discovery today, vol. 11, pp. 334-341, 2006.

[42] S. Fields and O.-K. Song, “A novel genetic system to detect protein–protein interactions,”

Nature, vol. 340, pp. 245 - 246, 1989.

150

[43] J. E. Butler, “Solid Supports in Enzyme-Linked Immunosorbent Assay and Other Solid-Phase

Immunoassays,” Methods, vol. 22, pp. 4-23, 2000.

[44] W. Kusnezow and J. D. Hoheisel, “Solid supports for microarray immunoassay,” Journal of

Molecular Recognition, vol. 16, pp. 165-176, 2003.

[45] Y. H. Tan, B. Pandey, A. Sharma, J. Bhattarai and K. J. Steine, “Bioconjugation reactions for

covalent coupling of proteins to gold surfaces,” Global Journal of Biochemistry, vol. 3, pp. 1-21,

2012.

[46] P. Schaaf, V. Ball, P. Huetz, A. Elaissari, J.-P. Cazenave, J.-C. Voegel and P. Schaff, “Kinetics of

exchange processes in the adsorption of proteins on solid surfaces,” Proceedings of the National

Academy of Sciences, vol. 91, pp. 7330-7334, 1994.

[47] E. Lutanie, J.-C. Voegel, P. Schaaf, M. Freund, J.-P. Cazenave and A. Schmitt, “Competitive

adsorption of human immunoglobulin G and albumin: consequences for structure and reactivity

of the adsorbed layer,” Proceedings of the National Academy of Sciences, vol. 89, pp. 9890-

9894, 1992.

[48] F. Rusmini, Z. Zhong and J. Feijen, “Protein Immobilization Strategies for Protein Biochips,”

Biomacromolecules, vol. 8, p. 1775–1789, 2007.

[49] R. Fernandez-Lafuente, C. M. Rosell, V. Rodriguez, C. Santana, G. Soler, A. Bastida and J. M.

Guisan, “Preparation of activated supports containing low pK amino groups. A new tool for

protein immobilization via the carboxyl coupling method,” Enzyme and Microbial Technology,

vol. 15, pp. 546-550, 1993.

[50] I. A. Banerjee, L. Yu, R. I. MacCupsie and H. Matsui, “Thiolated Peptide Nanotube Assembly of

Arrays on Patterned Au Substrates,” Nanoletters, vol. 4, pp. 2437-2440, 2004.

[51] O. R. Bolduc and J.-F. Masson, “Monolayers of 3-mercaptopropyl-amino acid to reduce the

nonspecific adsorption of serum proteins on the surface of biosensors,” Langmuir, vol. 24, pp.

12085-12091, 2008.

[52] V. K. Yadavalli, J. G. Forbes and K. Wang, “Functionalized Self-Assembled Monolayers on

Ultraflat Gold as Platforms for Single Molecule Force Spectroscopy and Imaging,” Langmuir, vol.

22, pp. 6969-6976, 2006.

[53] A. Hirlekar Schmid, S. Stanca, M. S. Thakur, K. Ravindranathan Thampia and C. Raman Suri, “Site-

directed antibody immobilization on gold substrate for surface plasmon resonance sensors,”

Sensors and Actuators B, vol. 113, p. 297–303, 2006.

[54] R. G. Nuzzo and D. L. Allara, “Adsorption of Bifunctional Organic Disulfides on Gold Surfaces,”

Journal of the American Chemical Society, vol. 105, pp. 4481-4483, 1983.

[55] C. D. Bain, E. B. Troughton, Y.-T. Tao, J. Evall, G. M. Whitesides and R. Nuzzo, “Formation of

Monolayer Films by Spontaneous Assembly of Organic Thiols from Solution onto Gold,” Journal

of the American Chemical Society, vol. 111, pp. 321-335, 1989.

151

[56] H. A. Biebuyck, C. D. Bain and G. M. Whitesides, “Comparison of Organic Monolayers on

Polycrystalline Gold Spontaneously Assembled from Solutions Containing Dialkyl Disulfides or

Alkanethiols,” Langmuir, vol. 10, p. 1825–1831, 1994.

[57] C. D. Bain, H. A. Biebuyck and G. M. Whitesides, “Comparison of self-assembled monolayers on

gold: coadsorption of thiols and disulfides,” Langmuir, vol. 5, p. 723–727, 1989.

[58] J. Noh, T. Murase, K. Nakajima, H. Lee and M. Hara, “Nanoscopic Investigation of the Self-

Assembly Processes of Dialkyl Disulfides and Dialkyl Sulfides on Au(111),” The Journal of Physical

Chemistry B, vol. 104, p. 7411–7416, 2000.

[59] C. Jung, O. Dannenberger, Y. Xu, M. Buck and M. Grunze, “Self-Assembled Monolayers from

Organosulfur Compounds: A Comparison between Sulfides, Disulfides, and Thiols,” Langmuir,

vol. 14, p. 1103–1107, 1998.

[60] S. Frey, V. Stadler, K. Heister, W. Eck, M. Zharnikov, M. Grunze, B. Zeysing and A. Terfort,

“Structure of Thioaromatic Self-Assembled Monolayers on Gold and Silver,” Langmuir, vol. 17,

p. 2408–2415, 2001.

[61] P. E. Laibinis, G. M. Whitesides, D. L. Allara, Y. T. Tao, A. N. Parikh and R. G. Nuzzo, “Comparison

of the structures and wetting properties of self-assembled monolayers of n-alkanethiols on the

coinage metal surfaces, copper, silver, and gold,” Journal of the American Chemical Society, vol.

113, p. 7152–7167, 1991.

[62] P. Fenter, P. Eisenberger, J. Li, N. Camillone, S. Bernasek, G. Scoles, T. A. Ramanarayanan and K.

S. Liang, “Structure of octadecyl thiol self-assembled on the silver(111) surface: an

incommensurate monolayer,” Langmuir, vol. 7, p. 2013–2016, 1991.

[63] M. M. Walczak, C. Chung, S. M. Stole, C. A. Widrig and M. D. Porter, “Structure and interfacial

properties of spontaneously adsorbed n-alkanethiolate monolayers on evaporated silver

surfaces,” Journal of the American Chemical Society, vol. 113, p. 2370–2378, 1991.

[64] J. C. Love, D. B. Wolfe, R. Haasch, M. L. Chabinyc, K. E. Paul, G. M. Whitesides and R. G. Nuzzo,

“Formation and Structure of Self-Assembled Monolayers of Alkanethiolates on Palladium,”

Journal of the American Chemical Society, vol. 125, pp. 2597-2609, 2003.

[65] E. Li, S.-C. Chang and S. R. Williams, “Self-Assembly of Alkanethiol Molecules onto Platinum and

Platinum Oxide Surfaces,” Langmuir, vol. 19, pp. 6744-6749, 2003.

[66] N. Muskal, I. Turyan and D. Mandler, “Self-assembled monolayers on mercury surfaces,” Journal

of Electrocnalytical Chemistry, vol. 409, pp. 131-136, 1996.

[67] G. E. Poirier, “Characterization of Organosulfur Molecular Monolayers on Au(111) using

Scanning Tunneling Microscopy,” Chemical Reviews, vol. 97, p. 1117–1128, 1997.

[68] C. O'Dwyer, G. Gay, B. Viaris de Lesegno and J. Weiner, “The Nature of Alkanethiol Self-

Assembled Monolayer Adsorption on Sputtered Gold Substrates,” Langmuir, vol. 20, pp. 8172-

8182, 2004.

152

[69] A. Ulman, “Formation and Structure of Self-Assembled Monolayers,” Chemical Reviews, vol. 96,

pp. 1533-1554, 1996.

[70] F. Schreiber, “Structure and growth of self-assembling monolayers,” Progress in Surface Science,

vol. 65, pp. 151-257, 2000.

[71] R. Subramanian and V. Lakshminarayanan, “A study of kinetics of adsorption of alkanethiols on

gold using electrochemical impedance spectroscopy,” Electrochimica Acta, vol. 45, pp. 4501-

4509, 2000.

[72] M. Boncheva and H. Vogel, “Formation of Stable Polypeptide Monolayers at Interfaces:

Controlling Molecular Conformation and Orientation,” Biophysical Journal, vol. 73, pp. 1056-

1072, 1997.

[73] K. Park, J. M. Lee, Y. Jung, T. Habtemariam, A. W. Salah, C. D. Fermin and M. Kim, “Combination

of cysteine- and oligomerization domain-mediated protein immobilization on a surface plasmon

resonance (SPR) gold chip surface,” Analyst, vol. 136, pp. 2506-2511, 2011.

[74] P. Sejwal, S. K. Narasimhan, D. Prashar, D. Bandyopadhyay and Y.-Y. Luk, “Selective

Immobilization of Peptides Exclusively viaN-Terminus Cysteines by Water-Driven Reactions on

Surface,” The Journal of Organic Chemistry, vol. 74, pp. 6843-6846, 2009.

[75] J. J. Davis, C. M. Halliwell, H. A. O. Hill, G. W. Canters, M. C. van Amsterdam and M. P. Verbeet,

“Protein adsorption at a gold electrode studied by in situ scanning tunnelling microscopy,” New

Journal of Chemistry, pp. 1119-1123, 1998.

[76] T. Sakurai, S. Oka, A. Kubo, K. Nishiyama and I. Taniguchi, “Formation of oriented polypeptides

on Au(111) surface depends on the secondary structure controlled by peptide length,” Journal

of peptide Science, vol. 12, pp. 396-402, 2006.

[77] A. B. Steel, T. M. Herne and M. J. Tarlov, “Electrochemical Quantitation of DNA Immobilized on

Gold,” Analytical Chemistry, vol. 70, pp. 4670-4677, 1998.

[78] R. Levicky, T. M. Herne, M. J. Tarlov and S. K. Satija, “Using Self-Assembly To Control the

Structure of DNA Monolayers on Gold: A Neutron Reflectivity Study,” Journal of the American

Chemical Society, vol. 120, pp. 9787-9792, 1998.

[79] M. Steichen, Thèse de doctorat, Université Libre de Bruxelles, 2008.

[80] T. Doneux, A. De Rache, E. Triffaux, A. Meunier, M. Steichen and C. Buess-Herman,

“Optimization of the Probe Coverage in DNA Biosensors by a One-Step Coadsorption

Procedure,” ChemElectroChem, vol. 1, pp. 147-157, 2014.

[81] V. Brabec and V. Mornstein, “Electrochemical behaviour of proteins at graphite electrodes. I.

Electrooxidation of proteins as a new probe of protein structure and reactions,” Biochimica et

Biophysica Acta, vol. 625, pp. 43-50, 1980.

[82] V. Brabec and V. Mornstein, “Electrochemical behaviour of proteins at graphite electrodes: II.

Electrooxidation of amino acids,” Biophysical Chemistry, vol. 12, pp. 159-165, 1980.

153

[83] X. Cai, G. Rivas, P. Farias, H. Shiraishi, J. Wang and E. Palecek, “Potentiometric stripping analysis

of bioactive peptides at carbon electrodes down to subnanomolar concentrations,” Analytica

Chimica Acta, vol. 332, pp. 49-57, 1996.

[84] J. Y. Gerasimov and R. Y. Lai, “An electrochemical peptide-based biosensing platform for HIV

detection,” Chemical Communications, vol. 46, pp. 395-397, 2010.

[85] J. Y. Gerasimov and R. Y. Lai, “Design and characterization of an electrochemical peptide-based

sensor fabricated via "click" chemistry,” Chemical Communications, vol. 47, pp. 8688-8690,

2011.

[86] R. Li, H. Huang, L. Huang, Z. Lin, L. Guo, B. Qiu and G. Chen, “Electrochemical biosensor for

epidermal growth factor receptor detection with peptide ligand,” Electrochimica Acta, vol. 109,

pp. 233-237, 2013.

[87] H. Li, Y. Cao, X. Wu, Z. Ye and G. Li, “Peptide-based electrochemical biosensor for amyloid 1–42

soluble oligomer assay,” Talanta, vol. 93, pp. 358-363, 2012.

[88] M. Puiu, A. Idili, D. Moscone, F. Ricci and C. Bala, “A modular electrochemical peptide-based

sensor for antibody detection,” Chemical Communications, vol. 50, pp. 8962-8965, 2014.

[89] G. A. Orlowski and H.-B. Kraatz, “Evaluation of electron transfer rates in peptide films: Simplified

calculation and theory,” Electrochimica Acta, vol. 51, pp. 2934-2937, 2006.

[90] K. Sugawara, T. Kadoya, H. Kuramitz and S. Tanaka, “Voltammetric detection of ovalbumin using

a peptide labeled with an electractive compound,” Analytica Chimica Acta, vol. 834, pp. 37-44,

2014.

[91] P. Estrela, D. Paula, P. Li, S. D. Keighley, P. Migliorato, S. Laurenson and P. K. Ferrigno, “Label-

Free Detection of Protein interactions with peptide aptamers by open circuit potential

measurement,” Electrochimica Acta, vol. 53, pp. 6489-6496, 2008.

[92] P. Miao, J. Yin, L. Ning and X. Li, “Peptide-based electrochemical approach for apoptosis

evaluation,” Biosensors and Bioelectronics, vol. 62, pp. 97-101, 2014.

[93] L. Feng, L. Wu, J. Wang, J. Ren, D. Miyoshi, N. Sugimoto and X. Qu, “Detection of a prognostic

indicator in early-stage cancer using functionalized graphene-based peptide sensors,”

Advanced Materials, vol. 24, pp. 125-131, 2012.

[94] K. Kerman, H. Song, J. S. Ducan, D. W. Litchfield and H.-B. Kraatz, “Peptide Biosensors for the

Electrochemical Measurement of Protein Kinase Activity,” Analytical Chemistry, vol. 80, pp.

9395-9401, 2008.

[95] S. Martic, M. Labib and H.-B. Kraatz, “On chip electrochemical detection of sarcoma protein

kinase and HIV-1 reverse transcriptase,” Talanta, vol. 85, pp. 2430-2436, 2011.

[96] J. L. Shepherd, A. Kell, E. Chung, C. W. Sinclair, M. S. Workentin and D. Bizzotto, “Selective

Reductive Desorption of a SAM-Coated Gold Electrode Revealed Using Fluorescence

Microscopy,” Journal of the American Chemical Society, vol. 126, pp. 8329-8335, 2004.

154

[97] J. L. Shepherd and D. Bizzotto, “Characterization of Mixed Alcohol Monolayers Adsorbed onto

a Au(111) Electrode Using Electro-fluorescence Microscopy,” Langmuir, vol. 22, pp. 4869-4876,

2006.

[98] A. Musgrove, A. Kell and D. Bizzotto, “Fluorescence Imaging of the Oxidative Desorption of a

BODIPY-Alkyl-Thiol Monolayer Coated Au Bead,” Langmuir, vol. 24, pp. 7881-7888, 2008.

[99] J. N. Murphy, A. K. Chang, H.-Z. Yu and D. Bizzotto, “On the Nature of DNA Self-Assembled

Monolayers on Au: Measuring Surface Heterogeneity with Electrochemical in Situ Fluorescence

Microscopy,” Journal of the American Chemical Society, vol. 131, pp. 4042-4050, 2009.

[100] J. R. Casanova-Moreno and D. Bizzotto, “Electrochemistry and in situ fluorescence microscopy

of octadecanol layers doped with a BODIPY-labeld phospholipid: Investigating an adsorbed

heterogeneous layer,” Journal of Electroanalytical Chemistry, vol. 649, pp. 126-135, 2010.

[101] E. A. Josephs and T. Ye, “A Single-Molecule View of the Conformational Switching of DNA

tethered to a Gold Electrode,” Journal of the American Chemical Society, vol. 134, pp. 10021-

10030, 2012.

[102] E. A. Josephs and T. Ye, “Electric-Field Dependent Conformations of Single DNA Molecules on a

Model Biosensor Surface,” Nanoletters, vol. 12, pp. 5255-5261, 2012.

[103] E. A. Josephs and T. Ye, “Nanoscale Spatial Distribution of Thiolated DNA on Model Nucleic Acid

Sensor Surfaces,” ACS Nano, vol. 7, pp. 3653-3660, 2013.

[104] A. Meunier, E. Triffaux, D. Bizzotto, C. Buess-Herman and T. Doneux, “In Situ Fluorescence

Microscopy Study of the Interfacial Inhomogeneity of DNA Mixed Self-Assembled Monolayers

at Gold Electrodes,” ChemElectroChem, vol. 2, pp. 434-442, 2015.

[105] O. R. Bolduc, D. Correia-Ledo and J.-F. Masson, “Electroformation of peptide self-assembled

monolayers on gold,” Langmuir, vol. 28, pp. 22-26, 2012.

[106] O. R. Bolduc, J. N. Pelletier and J.-F. Masson, “SPR biosensing in crude serum using ultralow

fouling binary patterned peptide SAM,” Analytical Chemistry, vol. 82, pp. 3699-3706, 2010.

[107] K. Nowinski, F. Sun, D. White, J. Keefe and S. Jiang, “Sequence, structure, and function of peptide

self-assembled monolayers,” Journal of the American Chemical Society, vol. 134, pp. 6000-6005,

2012.

[108] S. Chen, Z. Cao and S. Jiang, “Ultra-low fouling peptide surfaces derived from natural amino

acids,” Biomaterials, vol. 30, pp. 5892-5896, 2009.

[109] E. Gatto, A. Prochetta, M. Scarselli, M. De Crescenzi, F. Formaggio, C. Toniolo and M. Venanzi,

“Playing with Peptides: How to Build a Supramolecular Peptide Nanostructure by Exploiting

Helix Helix Macrodipole Interactions,” Langmuir, vol. 28, pp. 2817-2826, 2012.

[110] E. Gatto and M. Venanzi, “Self-assembled monolayers formed by helical peptide buiding blocks:

a new tool for bioinspired nanotechnology,” Polymer Journal, pp. 1-13, 2013.

155

[111] K. Fujita, N. Bunjes, K. Nakajima, M. Hara, H. Sasabe and W. Knoll, “Macrodipole Interaction of

Helical Peptides in a Self-Assembled Monolayer on Gold Substrate,” Langmuir, vol. 14, pp. 6167-

6172, 1998.

[112] L. Duchesne, G. Wells, D. G. Fernig, S. A. Harris and R. Levy, “Supramolecular Domains in Mixed

Peptide Self-Assembled Monolayers on Gold Nanoparticles,” ChemBioChem, vol. 9, pp. 2127-

2134, 2008.

[113] S. M. Morris, “A role for p53 in the frequency and mechanism of mutation,” Mutation Research,

vol. 511, pp. 45-62, 2002.

[114] J. G. Teodoro, S. K. Evans and M. R. Green, “Inhibition of tumor angiogenesis by p53: a new role

for the guardian of the genome,” Journal of Molecular Medicine, vol. 85, pp. 1175-1186, 2007.

[115] J. S. Fridman and S. W. Lowe, “Control of apoptosis by p53,” Oncogene, vol. 22, pp. 9030-9040,

2003.

[116] K. H. Vousden and X. Lu, “Live or let die: the cell's response to p53,” Nature Reviews Cancer, vol.

2, pp. 594-604, 2002.

[117] A. B. Deleo, G. Jay, E. Appella, G. C. Dubois, L. W. Law and L. J. Old, “Detection of a

transformation-related antigen in chemically induced sarcomas and other transformed cells of

the mouse,” Proceedings of the National Academy of Sciences, vol. 76, pp. 2420-2424, 1979.

[118] D. P. Lane and L. V. Crawford, “T antigen bound to a host protein in SV40-transformed cells,”

Nature, vol. 278, pp. 261-263, 1979.

[119] D. I. Linzer and A. J. Levine, “Characterization of a 54 kDalton cellular SV40 tumor antigen

present in SV40-transformed cells and uninfected embryonal carcinoma cells,” Cell, vol. 17, pp.

43-52, 1979.

[120] M. S. Greenblatt, W. P. Bennett, M. Hollstein and C. C. Harris, “Mutations in the p53 Tumor

Suppressor Gene: Clues to Cancer Etiology and Molecular Pathogenesis,” Cancer Research, vol.

54, pp. 4855-4878, 1994.

[121] J. Momand, G. P. Zambetti, D. C. Olson, D. George and A. J. Levine, “The mdm-2 oncogene

product forms a complex with the p53 protein and inhibits p53-mediated transactivation,” Cell,

vol. 69, pp. 1237-1245, 1992.

[122] S. S. Fakharzadeh, S. P. Trusko and D. L. George, “Tumorogenic potential associated with

enhanced expression of a gene that is amplified in a mouse tumor cell line,” The EMBO Journal,

vol. 10, pp. 1565-1569, 1991.

[123] J. Oliner, K. Kinzler, P. Meltzer, D. George and B. Vogelstein, “Amplification of a gene encoding

a p53-associated protein in human sarcomas,” Nature, vol. 358, pp. 80-83, 1992.

[124] C. Cordon-Cardo, E. Latres, M. Drobujak, M. R. Oliva, D. Pollack, J. M. Woodruff, V. Marechal, J.

Chen, M. F. Brennan and A. J. Levine, “Molecular Abnormalities of mdm2 and p53 Genes in

Adult Soft Tissue Sarcomas',” Cancer Research, vol. 54, pp. 794-799, 1994.

156

[125] F. S. Leach, T. Tokino, P. Meltzer, M. Burrell, J. D. Oliner, S. Smith, D. E. Hill, D. Sidransky, K. W.

Kinzler and B. Vogelstein, “p53 Mutation and MDM2 Amplification in Human Soft Tissue

Sarcomas,” Cancer Research, pp. 2231-2234, 1993.

[126] G. Reifenberger, L. Liu, K. Ichimura, E. E. Schmidt and V. P. Collins, “Amplification and

Overexpression of the MDM2 Gene in a Subset of Human Malignant Gliomas without p53

Mutations,” Cancer Research, vol. 53, pp. 2736-2739, 1993.

[127] C. Bueso-Ramos, Y. Yang, E. deLeon, P. McCown, S. Stass and M. Albitar, “The Human MDM2

Oncogene is Overexpressed in Leukemias,” Blood, vol. 82, pp. 2617-2623, 1993.

[128] A. Marchetti, F. Buttitta, S. Girlando, P. Dalla Palma, S. Pellegrini, P. Fina, C. Doglioni, G.

Bevilacqua and M. Barbareschi, “MDM2 gene alterations and MDM2 protein expression in

breast carcinomas,” The Journal of Pathology, vol. 175, pp. 31-38, 1995.

[129] I. Shibagaki, H. Tanaka, Y. Shimada, T. Wagata, M. Ikenaga, M. Imamura and K. Ishizaki, “p53

mutation, murine double minute 2 amplification, and human papillomavirus infection are

frequently involved but not associated with each other in esophageal squamous cell

carcinoma,” Clinical Cancer Research, vol. 1, pp. 769-773, 1995.

[130] J. Momand, H.-H. Wu and G. Dasgupta, “MDM2— master regulator of the p53 tumor suppressor

protein,” Gene, vol. 242, pp. 15-29, 2000.

[131] T. Juven-Gershon and M. Oren, “Mdm2: The Ups and Downs,” Molecular Medicine, vol. 5, pp.

71-83, 2000.

[132] J. Momand, D. Jung, S. Wilczynski and J. Niland, “The MDM2 gene amplification database,”

Nucleic Acids Research, vol. 26, pp. 3453-3459, 1998.

[133] S. Daujat, H. Neel and J. Piette, “MDM2:life without p53,” Trends in Genetics, vol. 17, pp. 459-

464, 2001.

[134] D. Michael and M. Oren, “The p53-Mdm2 module and the ubiquitin system,” Seminars in Cancer

Biology, vol. 13, pp. 49-58, 2003.

[135] E. Pennisi, “Filling In the Blanks in the p53 Protein Structure,” Science, vol. 274, pp. 921-922,

1996.

[136] S. Wang, Y. Zhao, D. Bernard, A. Aguilar and S. Kumar, “Targeting the MDM2-p53 Protein-

Protein Interaction for New Cancer Therapeutics,” in Topics in Medicinal Chemistry: Protein-

Protein Interactions, vol. 8, Berlin Heidelberg, Springer, 2012, pp. 57-80.

[137] P. H. Kussie, S. Gorina, V. Marechal, B. Elenbaas, J. Moreau, A. J. Levine and N. P. Pavletich,

“Structure of the MDM2 Oncoprotein Bound to the p53 Tumor Suppressor Transactivation

Domain,” Science, vol. 274, pp. 948-953, 1996.

[138] M. A. McCoy, J. J. Gesell, M. M. Senior and D. F. Wyss, “Lid to the p53-Binding Domain of Human

Mdm2: Implications for p53 Regulation,” Proceedings of the National Academy of Sciences, vol.

100, pp. 1645-1648, 2003.

157

[139] N. Magnasco Nichols and K. Shive Matthews, “p53 Unfolding Detected by CD but Not by

Tryptophan Fluorescence,” Biochemical and Biophysical Research Communications, vol. 288,

pp. 111-115, 2001.

[140] T. M. Webber, A. C. Allen, W. K. Ma, R. G. Molloy and C. N. Kettelkamp, “Conformational

detection of p53's oligomeric state by FlAsH Fluorescence,” Biochemical and Biophysical

Research Communications, vol. 384, pp. 66-70, 2009.

[141] M. Wallace, E. Worall, S. Pettersson, T. R. Hupp and K. L. Ball, “Dual-Site Regulation of MDM2

E3-Ubiquitin Ligase Activity,” Molecular Cell, vol. 23, pp. 251-263, 2006.

[142] L. M. Espinoza-Fonseca and J. Garcia-Machorro, “Aromatic-aromatic interactions in the

formation of the MDM2-p53 complex,” Biochemical and Biophysical Research Communications,

vol. 370, pp. 547-551, 2008.

[143] Z. Liu, E. T. Olejniczak and S. W. Fesik, “Over-expression of the human MDM2 p53 binding

domain by fusion to a p53 transactivation domain,” Protein Expression and Purification, vol. 37,

pp. 493-498, 2004.

[144] M. Wells, H. Tidow, T. J. Rutherford, P. Markwick, J. Ringkjobing, E. Mylonas, D. I. Svergun, M.

Blackledge and A. F. Fersht, “Structure of Tumor Suppressor p53 and Its Intrinsically Disorderd

N-Terminal Transactivation Domain,” Proceedings of the National Academy of Sciences, vol.

105, pp. 5762-5767, 2008.

[145] J. D. Oliner, J. A. Pietenpol, S. Thiagalingam, J. Gyuris, K. W. Kinzler and B. Vogelstein,

“Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53,” Nature, vol.

362, pp. 857-860, 1993.

[146] J. Lin, J. Chen, B. Elenbaas and A. J. Levine , “Several hydrophobic amino acids in the p53 amino-

terminal domain are required for transcriptional activation, binding to mdm-2 and the

adenovirus 5 E1B 55-kD protein,” Genes & Development, vol. 8, pp. 1235-1246, 1994.

[147] S. Picksley, B. Vojtesek, A. Sparks and D. P. Lane, “Immunochemical analysis of the interaction

of p53 with MDM2;--fine mapping of the MDM2 binding site on p53 using synthetic peptides,”

Oncogene, vol. 9, pp. 2523-2529, 1994.

[148] R. Zhang and H. Wang, “MDM2 Oncogene as a Novel Target for Human Cancer Therapy,”

Current Pharmaceutical Design, vol. 6, pp. 393-416, 2000.

[149] E. Rayburn, R. Zhang, J. He and H. Wang, “MDM2 and Human Malignancies: Expression, Clinical

Pathology, Prognostic Markers, and Implications for Chemotherapy,” Current Cancer Drug

Targets, vol. 5, pp. 27-41, 2005.

[150] Y. Takahashi, Y. Oda, K.-i. Kawaguchi, S. Tamiya, H. Yamamoto, S. Suita and M. Tsuneyoshi,

“Altered expression and molecular abnormalities of cell-cycle-regulatory proteins in

rhabdomyosarcoma,” Modern Pathology, vol. 17, pp. 660-669, 2004.

[151] M. Ladanyi, R. Lewis, S. C. Jhanwar, W. Gerald, A. G. Huvos and J. H. Healey, “MDM2 and CDK4

Gene Amplification in Ewing's Sarcoma,” The Journal of Pathology, vol. 175, pp. 211-217, 1995.

158

[152] J. M. Gudas, H. Nguyen, R. C. Klein, D. Katayose, P. Seth and K. H. Cowan, “Differential Expression

of Multiple MDM2 Messenger RNAs and Proteins in Normal and Tumorigenic Breast Epithelial

Cells,” Clinical Cancer Research, vol. 1, pp. 71-80, 1995.

[153] J. Weaver, P. Rao, J. R. Goldblum, M. J. Joyce, S. L. Turner, A. J. Lazar, D. Lopez-Terada, R. R.

Tubbs and B. P. Rubin, “Can MDM2 analytical tests performed on core needle biopsy be relied

upon to diagnose well-differentiated liposarcoma?,” Modern Pathology, vol. 23, pp. 1301-1306,

2010.

[154] Y. A. Valentin-Vega, J. A. Barboza, G. P. Chau, A. K. El-Naggar and G. Lozano, “High levels of the

p53 inhibitor MDM4 in head and neck squamous carcinomas,” Human Pathology, vol. 38, pp.

1553-1562, 2007.

[155] R. Elshafey, C. Tlili, A. Abulrob, A. C. Tavares and M. Zourob, “Label-free impedimetric

immunosensor for ultrasensitive detection of cancer marker Murine double minute 2 in brain

tissue,” Biosensors and Bioelectronics, vol. 39, pp. 220-225, 2013.

[156] H. Li, H. Xie, Y. Huang, B. Bo, X. Zhu, Y. Shu and G. Li, “Highly sensitive protein detection based

on a novel probe with catalytic activity combined with a signal amplification strategy: assay of

MDM2 for cancer staging,” Chemical Communications, vol. 49, pp. 9848-9850, 2013.

[157] A. Hamelin, “Cyclic voltammetry at gold single-crystal surfaces. Part 1. Behaviour at low-index

faces,” Journal of Electroanalytical Chemistry, vol. 407, pp. 1-11, 1996.

[158] A. Hamelin, “Cyclic voltammetry at gold single-crystal surfaces. Part 2. Behaviour of high-index

faces,” Journal of Electroanalytical Chemistry, vol. 407, pp. 13-21, 1996.

[159] O. Schon, A. Friedler, M. Bycroft, S. M. Freund and A. R. Fersht, “Molecular Mechanism of the

Interaction between MDM2 and p53,” Journal of Molecular Biology, vol. 323, pp. 491-501, 2002.

[160] K. Kusmierczyk, M. Lukaszewski, Z. Rogulski, H. Siwek, J. Kotowski and A. Czerwinski,

“Electrochemical Behavior of Pt-Au alloys,” Polish Journal of Chemistry, vol. 76, pp. 607-618,

2002.

[161] Southampton Electrochemistry Group, Instrumental Methods in Electrochemistry, Chichester:

Horwood Publishing,Chemical science series, 2001.

[162] A. J. Bard and L. R. Faulkner, Electrochemical Methods: Fundamentals and Applications, 2nd ed.,

New York: John Wiley &Sonc, Inc., 2001.

[163] M. E. Orazem and B. Tribollet, Electrochemical Impedance Spectroscopy, Hoboken: John Wiley

& Sons, Inc., 2008.

[164] A. Janshoff, H.-J. Galla and C. Steinem, “Piezoelectric Mass-Sensing Devices as Biosensors-An

Alternative to Optical Biosensors?,” Angewandte Chemie International Edition, vol. 39, pp.

4004-4032, 2000.

[165] U. Rant, K. Arinaga, S. Fujita, N. Yokoyama, G. Abstreiter and M. Tornow, “Structural Properties

of Oligonucleotide Monolayers on Gold Surface Probed by Fluorescence Investigations,”

Langmuir, vol. 20, pp. 10086-10092, 2004.

159

[166] H. Finklea, in Electroanalytical Chemistry, vol. 19, A. Bard and I. Rubinstein, Eds., 1996, pp. 109-

335.

[167] M. A. Coleman, V. H. Lao, B. W. Segelke and P. T. Beernink, “High-Throughput, Fluorescence-

Based Screening for Soluble Protein Expression,” Journal of Proteome Research, vol. 3, p. 1024–

1032, 2004.

[168] D. Finnskog, A. Ressine, T. Laurell and G. Marko-Varga, “Integrated Protein Microchip Assay with

Dual Fluorescent- and MALDI Read-Out,” Journal of Proteome Research, vol. 3, p. 988–994,

2004.

[169] G. Hui, “UPA, a universal protein array system for quantitative detection of protein–protein,

protein–DNA, protein–RNA and protein–ligand interactions,” Nucleic Acids Research, vol. 28, p.

e2, 2000.

[170] K.-Y. Tomizaki, K. Usui and H. Mihara, “Protein-Detecting Microarrays: Current

Accomplishments and Requirements,” ChemBioChem, vol. 6, pp. 782-799, 2005.

[171] H.-Z. Yu, C.-Y. Luo, C. G. Sankar and S. Dipankar, “Voltammetric Procedure for Examining DNA-

Modified Surfaces: Quantitation, Cationic Binding Activity, and Electron-Transfer Kinetics,”

Analytical Chemistry, vol. 75, pp. 3902-3907, 2003.

[172] L. Su, C. G. Sankar, D. Sen and H.-Z. Yu, “Kinetics of Ion-Exchange Binding of Redox Metal Cations

to Thiolate-DNA Monolayers on Gold,” Analytical Chemistry, vol. 76, pp. 5953-5959, 2004.

[173] M. Steichen, T. Doneux and C. Buess-Herman, “On the adsorption of hexaammineruthenium

(III) at anionic self-assembled monolayers,” Electrochimica Acta, vol. 53, p. 6202–6208, 2008.

[174] A. J. Bard and L. R. Faulkner, “Chapter 14. Electroactive Layers and Modified Electrodes,” in

Electrochemical Methods: Fundamentals and Applications, 2nd Edition ed., New York, John

Wiley &Sonc, Inc., 2001, pp. 580-631.

[175] A. B. Steel, T. M. Herne and M. J. Tarlov, “Electrostatic Interactions of Redox Cations with

Surface-Immobilized and Solution DNA,” Bioconjugate Chemistry, vol. 10, pp. 419-423, 1999.

[176] K. Arinaga, U. Rant, M. Tornow, S. Fujita, G. Abstreiter and N. Yokoyama, “The Role of Surface

Charging during the Coadsorption of Mercaptohexanol to DNA Layers on Gold: Direct

Observation of Desorption and Layer Reorientation,” Langmuir, vol. 22, pp. 5560-5562, 2006.

[177] D. M. Ceres, A. K. Udit, H. D. Hill, M. G. Hill and J. K. Barton, “Differential Ionic Permeation of

DNA-Modified Electrodes,” Journal of Physical Chemistry B, vol. 111, pp. 663-668, 2007.

[178] H. Lee, K. H. Mok, R. Muhandiram, K.-H. Park, J.-E. Suk, D.-H. Kim, J. Chang, Y. C. Sung, K. Y. Choi,

H. Kyou-Hoon and K.-H. Han, “Local structural elements in the mostly unstructured

transcriptional activation domain of human p53,” The Journal of biological chemistry, vol. 275,

pp. 29426-29432, 2000.

[179] J. Y. Chen, M. Li, L. S. Penn and J. Xi, “Real-Time and Label-Free Detection of Cellular Response

to Signaling Mediated by Distinct Subclasses of Epidermal Growth Factor Receptors,” Analytical

Chemistry, vol. 83, pp. 3141-3146, 2011.

160

[180] H. Choi and S.-J. Choi, “Detection of Edwardsiella tarda by fluorometric or biosensor methods

using peptide ligand,” Analytical Biochemistry, vol. 421, pp. 152-157, 2012.

[181] T. Hianik, V. Ostatna , Z. Zajacova, E. Stoikova and G. Evtugyn, “Detection of aptamer-protein

interactions using QCM and electrochemical indicator methods,” Bioorganic & Medicinal

Chemistry Letters, vol. 15, pp. 291-295, 2005.

[182] T. Hianik, V. Ostatna and Z. Zajacova, “The study of the binding of globular protein to DNA using

mass detection and electrochemical indicator methods,” Journal of Electroanalytical Chemistry,

vol. 564, pp. 19-24, 2004.

[183] Y. Xiao and S. N. Isaacs, “Enzyme-linked immunosorbent assay (ELISA) and blocking with bovine

serum albumin (BSA)—not all BSAs are alike,” Journal of Immunological Methods, vol. 384, pp.

148-151, 2012.

[184] Y. Lin, J. Wang, L.-J. Wan and X.-H. Fang, “Study of fibrinogen adsorption on self-assembled

monolayers on Au(111) by atomic force microscopy,” Ultramicroscopy, vol. 105, p. 129–136,

2005.

[185] K. K. Kanazawa and J. G. Gordon, “The oscillation frequency of a quartz resonator in contact

with liquid,” Analytica Chimica Acta, vol. 175, pp. 99-105, 1985.

[186] K. K. Kanazawa and J. G. Gordon, “Frequency of a quartz microbalance in contact with liquid,”

Analytical Chemistry, vol. 57, pp. 1770-1771, 1985.

[187] S. Bruckenstein and M. Shay, “Experimental aspects of the use of the quartz crystal

microbalance in solution,” Electrochimica Acta, vol. 30, pp. 1295-1300, 1985.

[188] [Online]. Available: http://www.biocompare.com/pfu/110627/soids/2-

2010/ELISA_Kit/ELISA_MDM2. [Accessed 16 06 2015].

[189] D. A. Skoog, J. F. Holler and T. A. Nieman, Principles of Instrumental Analysis, 5th ed., H. B. &.

Company, Ed.

[190] B. Valeur and M. N. Berberan-Santos, Molecular Fluorescence Principle and Applications, 2nd

ed., Wiley-VCH, 2012.

[191] S. S. Vogel, B. W. van der Meer and P. S. Blank, “Estimating the distance separating fluorescent

protein FRET pairs,” Methods, vol. 66, pp. 131-138, 2014.

[192] L. Stryer and R. Haugland, “Energy transfer: a spectroscopic ruler,” Proceedings of the National

Academy of Sciences, vol. 58, pp. 719-726, 1967.

[193] R. R. Chance, A. Prock and R. Silbey, “Molecular Fluorescence and Energy Transfer Near

Interfaces,” in Advances in Chemical Physics, vol. 37, I. Prygogine and S. A. Rice, Eds., Hoboken,

John Wiley & Sons, Inc., 1978.

[194] H. Kuhn, “Classical Aspects of Energy Transfer in Molecular Systems,” The Journal of Chemical

Physics, vol. 53, pp. 101-108, 1970.

161

[195] U. Rant, K. Arinaga, S. Fujita, N. Yokoyama, G. Abstreiter and M. Tornow, “Dynamic Electrical

Switching of DNA Layers on a Metal Surface,” Nanoletters, vol. 4, pp. 2441-2445, 2004.

[196] S. Takeishi, U. Rant, T. Fujiwara, K. Buchholz, T. Usuki, K. Arinaga, K. Takemoto, Y. Yamaguchi,

M. Tornow, S. Fujita, G. Abstreiter and N. Yokoyama, “Observation of electrostatically released

DNA from gold electrodes with controlled treshold voltages,” Journal of Chemical Physics, vol.

120, pp. 5501-5504, 2004.

[197] [Online]. Available:

http://www.microscopyu.com/articles/fluorescence/filtercubes/filterindex.html. [Accessed 05

06 2015].

[198] [Online]. Available: http://www.microscopyu.com/tutorials/flash/spectralprofiles/index.html.

[Accessed 08 06 2015].

[199] [Online]. Available: http://www.rocketmime.com/astronomy/Telescope/ResolvingPower.html.

[Accessed 08 06 2015].

[200] [Online]. Available: http://zeiss-campus.magnet.fsu.edu/articles/basics/resolution.html.

[Accessed 08 06 2015].

[201] [Online]. Available: http://rsb.info.nih.gov/ij/. [Accessed 16 06 2015].

[202] J. C. Russ, The Image Processing Handbook, 6th ed., Boca Raton: CRC Press Taylor & Francis

Group, 2011.

[203] N. Otsu, “A Threshold Selection Method from Grey-Level Histograms,” IEEE Transactions on

Systems, Man, and Cybernetics, vol. SMC9, pp. 62-66, 1979.

162

Date post:	25-Mar-2020
Category:	Documents
Upload:	others
View:	2 times
Download:	0 times

Interfacial study of a sensing platform for MDM2, based on ... · Léo, j’ai toujours pu compter...

Documents