1

Table of contents

La mise au point d'outils performants pour détecter rapidement et à des états précoces ces deux pathologies est un dé fi auquel je me suis attaché e durant ma thè se. De tels exemples ont é té utilisé s pour dé montrer que par la suite ces capteurs d'ADN pourraient ê tre gé né ralisé s et é tendues à toutes autres maladies infectieuses.

La particularité des capteurs électrochimiques que j'ai développés durant ma thèse, repose sur le dé veloppement de nouveaux nanomaté riaux associant des porphyrines fonctionnalisé es à des feuilles de graphè ne, sur lesquels sont immobilisés les brins d'ADN d'intérêts. Cet assemblage moléculaire constitue la base du transducteur é lectrochimique, qui comme nous l'avons démontré, offre des propriétés très intéressantes aux capteurs en termes de gamme dynamique de dé tection, de limite de dé tection, de sensibilité et de sé lectivité . La plus grande partie de mon travail de thèse a été de mettre au point et d'optimiser la synthèse de ces maté riaux hybrides pour offrir les meilleures performances aux capteurs d'ADN à visés thé rapeutiques.

Ce manuscrit est constitué de deux parties. Une premiè re partie bibliographique introduit les capteurs d'ADN é lectrochimiques en gé né ral (avec ou sans marquage) ainsi que les diffé rents transducteurs é lectrochimiques utilisé s dans ce type de capteurs : les polymè res organiques conducteurs (notamment le polypyrrole), les nanoparticules et associé es et les maté riaux à base de carbone (le fullerè ne et les nanotubes de carbone). Cette partie pré sente é galement l'état de l'art sur le graphè ne incluant les diffé rents types de graphè ne et leurs approches de synthèse. J'expose ensuite les applications du graphène dans les capteurs d'ADN, les différentes approches d'immobilisation des brins d'ADN sur la surface du graphène et quelques exemples d'applications associant le graphène réduit à différents maté riaux (comme les nanoparticules, les polymè res conducteurs et des composé s aromatiques). Enfin, un dernier chapitre est consacré à la pré sentation des porphyrines, leur propriété électrochimique, leur synthèse suivi de quelques exemples d'application dans les capteurs associé s au graphè ne.

La deuxiè me partie du manuscrit pré sente mes ré sultats, divisé s en quatre chapitres qui dé crivent les diffé rents maté riaux hybrides é laboré s durant ma thè se et leur inté gration au sein de capteurs d'ADN. Ces chapitres sont 1) la synthè se et la caracté risation, par plusieurs techniques physico-chimiques des diffé rents nanomaté riaux é laboré s à base de graphè ne ré duit (RGO) et de porphyrines fonctionnalisé es, 2) le développement d'un capteur d'ADN à base de RGO et d'une porphyrine de manganèse, 3) étude de l'effet du nombre des fonctions acides carboxyliques de la porphyrine sur les performances du capteur à base de RGO et 4) é laboration d'un capteur auto-assemblé .

Synthè se et proprié té s des nanomaté riaux.

Dans cette partie, je pré sente tout d'abord, la synthè se de diffé rentes porphyrines utilisé es : la té traphé nylporphyrine portant une fonction carboxylique en para d'un groupement phé nyl, la H2TPP-1CP et une té traphé nylporphyrine substitué e par quatre fonctions carboxyliques, H2TPP-4CP. Les fonctions COOH sont utilisées comme point d'ancrage de l'ADN selon un couplage peptidique entre une fonction amine de l'ADN en position terminale et cette fonction COOH de la porphyrine. La té traphé nylporphyrine H2TPP-1CP est mé tallé e par un mé tal é lectroactif, le manganè se, (MnTPP-1CP) pour des é tudes complé mentaires. En effet, l'intérêt de préparer un tel complexe a permis la caractérisation du maté riau formé sur la surface en suivant le signal redox intense et ré versible du complexe de manganè se (III). Une straté gie de synthè se pour chaque molé cule est dé veloppé e et les molé cules sont correctement caractérisées selon les techniques d'analyses usuelles (RMN, SM, UV). Le graphè ne a é té ensuite pré paré selon diffé rents protocoles opé ratoires et caracté risé par XPS et Raman. Les diffé rentes qualité s de graphè ne obtenues sont comparé es et des é tudes é lectrochimiques ont dé montré que le graphè ne oxydé ré duit chimiquement (CRGO) offrait les meilleurs proprié té s en terme de conductivité et donc de transfert de charge, né cessaires pour la bonne mise en oeuvre du capteur électrochimique d'ADN.

Par la suite, les maté riaux hybrides sont pré paré s et dé posé s sur l'électrode de carbone soit en formant successivement une couche de graphè ne puis de porphyrines soit en pré formant le maté riau en solution. Chaque maté riau a é té caracté risé par microscopie é lectronique à balayage (MEB), spectroscopie de photoé lectrons aux rayons X (XPS), par spectrophotomé trie infrarouge à Transformé e de Fourier (FT-IR) et par é lectrochimie. Ces é tudes ont fait ressortir plusieurs avantages sur les nano hybrides. La quantité de porphyrine immobilisée sur la surface de l'électrode est accrue en raison de la grande surface d'adsorption du graphène. La structure conjuguée du graphène CRGO permet une interaction forte et stable avec les té traphé nylporphyrines par des interactions de type π-π. Ce mode d'interaction ne perturbe pas la structure é lectronique intrinsè que du CRGO et lui permet ainsi de conserver toutes ses proprié té s de conductivité et de transfert de charge.

L'augmentation des charges négatives sur la porphyrine permet une meilleure dispersion des feuilles de nano-hybrides sur la surface de l'électrode.

Une étude approfondie a été menée pour comprendre le mode d'association entre les porphyrines mono/ tetra carboxyliques avec le graphène. J'ai réalisé des expériences de microscopie à force atomique (AFM), spectroscopie UV-visibles et Raman. Ces é tudes ont fait ressortir que la té traphé nylporphyrine portant un groupement carboxylique viendrait interagir sur toute la surface du graphène et formerait plusieurs couches d'empilement. Dans le cas des porphyrines portant quatre fonctions acides carboxyliques, la pré sence des fonctions alcools et acides en pé riphé ries des feuilles de graphè ne privilé gierait des interactions aux extrémités plutôt qu'en surface du graphène.

Capteurs d'ADN à base de RGO et de porphyrine de manganè se.

L'objectif du chapitre II est une preuve de concept de notre approche par l'élaboration du premier capteur d'ADN basé sur un nanomaté riau associant du graphè ne (CRGO) à une porphyrine de manganè se mono fonctionnalisé e par une fonction carboxylique, comme point d'ancrage de l'ADN. Nous démontrerons que cette association entre le complexe et le graphè ne amé liore les proprié té s é lectriques et conductrices du maté riau lors de la dé tection électrochimique du brin d'ADN complé mentaire. Le manganè se (Mn) a é té choisi comme cation mé tallique, car le complexe formé possè de un signal é lectrochimique ré versible et intense qui a permis la caracté risation par voie é lectrochimique. La straté gie suivie pour la construction du capteur d'ADN repose sur un greffage covalent entre le brin d'ADN en solution et la fonction acide de la mé talloporphyrine, pré alablement immobilisé e sur l'électrode de carbone et le graphène.

Les conditions de greffage ont é té optimisé es par divers tests é lectrochimiques en faisant varier le rapport de concentration de MnTPP-1CP et CRGO, la concentration en ré actifs activé s (EDC et NHS), et le temps de greffage. Finalement, l'étape de couplage a été caracté risé e par diffé rentes techniques d'analyses telles que les mesures FT-IR, MEB, voltammé trie cyclique (VC) et impé dancemé trie (EIS). L'hybridation en présence de l'ADN complé mentaire a é té suivie par EIS dans une solution de [Fe(CN)6] 3-/ 4-à 5 mM et a permis de dé terminer une gamme dynamique de [100 aM-10 pM] et une limite de dé tection de 2×10 -16 M. Ce premier résultat s'est montré très encourageant et prometteur pour le développement de nouveaux outils à base de porphyrine et de graphè ne pour le diagnostic mé dical. Cependant, une é tude de la sé lectivité du capteur d'ADN par impé dancemé trie (EIS) a montré une variation du signal de 9% du capteur en présence d'une séquence non complémentaire contre 27% en présence d'une séquence modifiée au niveau d'une seule base de l'ADN. Ce résultat montre que ce nanomatériau hybride présente une surface d'adsorption non-spé cifique non négligeable. C'est pourquoi, nous avons cherché à améliorer cette propriété du capteur en diminuant ces interactions non souhaité es lors de la dé tection. Une des solutions proposé e a été d'augmenter le nombre de charges négatives sur la surface de l'électrode modifiée afin de limiter par répulsion de charges, les adsorptions des brins d'ADN, également chargés né gativement.

Ce travail d'optimisation du matériau a fait l'objet de la deuxième partie de ma thèse, par une é tude comparative entre deux porphyrines fonctionnalisé es.

Capteurs d'ADN à base de CRGO et de porphyrines polyfonctionnalisé es-Effet du nombre de fonctions acides carboxyliques sur les performances du capteur.

L'objectif du chapitre Ⅲ, s'inscrit dans la continuité du chapitre II, par une é tude comparative entre deux nanomaté riaux associant le graphè ne CRGO et une té traphé nylporphyrine fonctionnalisé e par un et quatre fonctions carboxyliques nommé s H2TPP-1CP/CRGO et H2TPP-4CP/CRGO respectivement. Le capteur d'ADN a été obtenu, dans chaque cas, en suivant les mê mes conditions que dans le chapitre II, en immergeant l'électrode, modifiée par le nanohybride, dans une solution de 1 µM contenant le brin d'ADN à greffer en présence d'agents activant (EDC et NHS). Les deux capteurs obtenus ssDNA/H2TPP-1CP/CRGO et ssDNA/H2TPP-4CP/CRGO respectivement, sont caracté risé s par voltammé trie par vague carré e (SWV) et impé dancemé trie (EIS). Les é tudes par impédance dans le PBS ont bien montré, dans les deux cas, l'immobilisation de l'ADN sur la surface, grâce à l'augmentation de la résistance du transfert d'électron (Rct). Cette augmentation s'explique par la présence de l'ADN sur la surface qui encombre la surface et rend le transfert d'électrons entre la surface et la solution plus difficile. L'épaisseur de la biocouche nanohybride (ssDNA/H2TPP-nCP/CRGO) a é té dé terminé e par impé dance et vaut 30 nm et 6 nm pour le capteur n=1 et n=4 respectivement. Ce ré sultat est en accord avec l'hypothèse émise précédemment, dans laquelle les porphyrines mono fonctionnalisé es (n=1) interagiraient plutôt sur la surface du graphè ne, contrairement aux porphyrines té trasubstitué es (n=4) qui se lieraient aux feuilles de graphè ne par leurs extré mité s. Ce ré sultat a été conforté par des études d'AFM. La réaction d'hybridation en pré sence du brin complémentaire a été suivie par voltammétrie à vague carrée (SWV) en présence d'une sonde redox neutre, la benzoquinone. Une gamme dynamique a é té obtenue pour les deux capteurs sur une gamme de concentration d'ADN du même ordre de grandeur et une limite de détection de l'ordre du femtomolaire. Par contre, il a été observé une différence lors de l'étude de la sélectivité de chacun de ces capteurs, en présence d'une concentration de 1 fmole du brin d'ADN non complémentaire. En effet, une variation de l'ordre de 30% de l'intensité du courant de ré duction de la benzoquinone a é té mesuré e par SWV pour le capteur ADN/H2TPP-1CP/CRGO contre seulement 15% pour le capteur chargé né gativement sur sa surface ADN/H2TPP-4CP/CRGO. Cette différence peut s'expliquer par la présence plus importante de charges négatives sur la surface de l'électrode à pH 7, pour le capteur ADN/H2TPP-4CP/CRGO, ré duisant ainsi par ré pulsions é lectroniques les adsorptions de l'ADN sur la surface.

Le capteur ADN/H2TPP-4CP/CRGO a é té ensuite utilisé pour dé tecter l'ADN du gè ne rpoB de Mycobacterium tuberculosis dans des é chantillons ré els de PCR. La sé quence d'ADN immobilisée est une sé quence complé mentaire du gè ne rpoB. Les ré sultats de dé tection et de sé lectivité sont de la mê me qualité que ceux obtenus lors de la dé tection du gène de l'hépatite B, avec une variation seulement de 10% du courant en présence du brin non complé mentaire. Ce ré sultat montre l'é norme potentiel de la plateforme H2TPP-4CP/CRGO pour la dé tection électrochimique d'agents pathogènes pour des applications de type diagnostique et thé rapeutique.

Par ailleurs, nous avons essayé de comprendre l'origine de ces adsorptions non spécifiques de l'ADN sur l'électrode. Nous avons émis l'hypothèse que ces interactions venaient de l'adsorption de l'ADN directement sur l'électrode de carbone. En effet, les feuilles de graphène ne recouvrant pas la totalité de l'électrode, des surfaces de carbone seraient ainsi disponibles pour l'adsorption des protéines ou de l'ADN. Cet aspect du travail est abordé dans le chapitre IV.

Capteurs d'ADN auto-assemblé s

Les protéines ou l'ADN sont connues pour s'adsorber sur de nombreuses surfaces dont le carbone. Plusieurs approches sont dé crites dans la litté rature pour limiter ou bloquer ces interactions (utilisations de protéines, de polyéthylène glycol..). L'une d'entre elles consiste à recouvrir la surface de carbone par une monocouche auto-assemblé e (SAM) de phé nylethylamine (β-PEA), couche qui pré sente des groupements phé nyles orienté s et perpendiculaires à la surface rendant celle-ci plus hydrophobe. Cette organisation devrait aussi amé liorer l'immobilisation du maté riau nanohybride par des interactions de type π-π entre le graphè ne et les groupes aromatiques terminaux de la couche SAM. Dans un premier temps, la formation de la couche de β-PEA a é té pré paré e et optimisé e par é lectrodé position du monomè re à diffé rentes concentrations (1mM, 2 mM et 4mM). Ces diffé rentes couches ont é té é tudié es et caracté risé es par des mesures de SWV, EIS et AFM. Puis nous avons immobilisé le maté riau hybride H2TPP-4CP/CRGO, qui s'est avéré être le plus performant dans le chapitre III. Ces é tudes ont montré que quelque soit la concentration en β-PEA, l'incorporation d'une telle monocouche entre l'électrode et le nanomatériau, H2TPP-4CP/CRGO, amé liore la conductivité de la couche en augmentant la ré versibilité du signal redox de la sonde é lectrochimique en solution (benzoquinone). Par ailleurs, une couche SAM obtenue par é lectrodé position de 2 mM de β-PEA conduit à une couche plus organisé e et une meilleure orientation des groupements phényles sur la surface de l'électrode. Cette organisation a permis ainsi une meilleure interaction du nanomaté riau H2TPP-4CP/CRGO avec la monocouche, rendant certainement plus accessible les fonctions carboxyliques à la surface. En effet, avec cette surface modifié e, nous avons obtenu un meilleur taux de recouvrement de l'ADN sur la couche (11 nmol.cm -2 ). La réaction d'hybridation a été ensuite é tudié e, par la technique SWV pour chacun des capteurs pré sentant des couches monoassemblé es diffé rentes (ADN/H2TPP-4CP/CRGO/1β-PEA, ADN/H2TPP-4CP/CRGO/2β-PEA et ADN/H2TPP-4CP/CRGO/4β-PEA). Nous avons observé lors de l'ajout croissant de concentration de l'ADN complémentaire de 1aM à 1 nM, une augmentation de l'intensité du courant de ré duction de la benzoquinone en hydroquinone pour les capteurs ADN/H2TPP- Ainsi au travers de mes travaux de thèse, j'ai pu élaborer un capteur d'ADN, à base de graphè ne et de porphyrine, qui pré sente un é norme potentiel pour la dé tection é lectrochimique d'agents pathogènes pour des applications de type diagnostique et thé rapeutique.

GENERAL INTRODUCTION

The project of my thesis consisted on the development of nanomaterials that are able to act as molecular transducers for the construction of DNA sensors, based on electrochemical Firstly, the hybrid nanomaterials which combined the features of both graphene (high conductivity and large specific surface area) and porphyrins (physical and chemical properties and electron transfer ability), such as tetraphenylporphyrin bearing one and four carboxylic groups named H2TPP-nCP(n=1, 4) and tetraphenylporphyrin incorporating manganese (III) (MnTPP-1CP) were successfully synthesized. The interaction between H2TPP-nCP(n=1, 4) and chemically reduced graphene oxide (CRGO) was studied in my thesis work. To investigate the characteristic properties of as-prepared nanomaterials, scanning electron microscopy (SEM), atomic force microscopy (AFM), UV-visible spectra, Fourier Transform infrared spectra (FT-IR), X-ray photoelectron spectra (XPS) and Raman spectroscopy were utilized.

The restored π-network of CRGO was expected to provide a good substrate for anchoring πconjugated molecules through π-π stacking interactions as non-covalent approach which does not disrupt the intrinsic electronic structure of the CRGO and retains the total conductivity of graphene. The porphyrins are able to incorporate many different metal ions for redox activities that can improve the ability of electron transfer and provide catalytic activity. The electrochemical properties was explored by electrochemical impedance spectroscopy (EIS)

and cyclic voltammetry (CV), which was expected to confirm that both the tight interaction exists between H2TPP-nCP(n=1,4) and CRGO, and the hybrid nanomaterial H2TPP-4CP/CRGO possesses more negative charge due to the presence of carboxylic groups on the surface introduced by attached porphyrins. The electrochemical process and reversibility monitored in neutral redox marker p-benzoquinone without charge ligands was to be tested to demonstrate that the hybrid nanomaterials possess high electroactivity with enhanced ks calculated by Nicolson model, compared to CRGO. In order to further investigate the characteristics and electrochemical properties of hybrid nanomaterial H2TPP-nCP/CRGO, a porphyrin of manganese (Mn) was to be utilized through the electrochemical test of the signal response of the Mn 3+ /Mn 2+ redox complex.

The purpose in chapter Ⅱ is to exploit a DNA sensor based on the hybrid nanomaterial MnTPP-1CP/CRGO which could improve the electrical properties and lead to a sensing layer for electrochemical DNA detection. The functionalization of phenyl ring with a carboxylic group allows the attachment of single-stranded amino DNA (ssDNA) on the complex through covalent interactions. The manganese (Mn) has been chosen as metallic cation, due to its reversible and electrochemical signal response that will allow the electrochemical characterization. The strategy followed for the construction of DNA sensor was to be were to be monitored by following the variation of the reduced current of redox couple pbenzoquinone/hydroquinone. On the basis of previously developed and optimized conditions, the electrochemical DNA sensor based on H2TPP-4CP/CRGO was expected to prepare and exhibite a broader linear range with a lower detection limit compared to ssDNA/H2TPP-1CP/CRGO platform. Furthermore, the DNA sensor was expected to be applied for sensing DNA of rpoB gene of Mycobacterium tuberculosis in real PCR samples. The elaborated DNA sensor was suitable for detection of sequences with wild-type samples, discriminating them from a single nucleotide polymorphism (SNP) T (TCG/TTG) with mutation. This shows enormous potential on this platform for further applications in pathogens diagnostic and therapeutic purpose.

According to some results before mentioned, the variation of the non-specific absorption value was still high and should be improved such as blocking the glassy carbon electrode with aromatic compounds to avoid any self-assembled adsorptions on the electrode. The Chapter Ⅳ will devote to eliminate non-specific absorptions.

The purpose of incorporating β-PEA layer in such DNA sensor was to underline homogeneous surface coverage of H2TPP-4CP/CRGO with preformed monolayers (M) with phenyl in end position. Such function could improve the immobilization of composite as followed, thus can be obtained by π-π staking interactions of composite formed with graphene and the terminal functional groups of SAM layer. In addition, such modification demonstrates its ability to prevent non-specific adsorptions of DNA on the electrode surface during the hybridization reaction. The formation of β-PEA layer was optimized by electrodeposition of monomer in different concentrations, followed by the immobilization of hybrid complex moieties H2TPP-4CP/CRGO and the grafting of ssDNA. All the steps of construction were to be studied and characterized by SWV, EIS measurements and AFM experiments. Via varying the concentration of β-PEA modified on the GCE surface, I expected to successfully obtain a homogeneous surface and allow to have a uniform covering of H2TPP-4CP/CRGO onto the surface of electrode with β-PEA, which was to make an electrochemical detection directly with an increase in redox marker signal called "signal on", which has many advantages compared to biosensors with decrease in signal "signal off". Detection of cDNA was investigated for each sensing layer. The superior performance of DNA sensor was expected to exhibit an excellent sensitivity with low limit detection and broad linear range for H2TPP-4CP/CRGO/-PEA platform. Moreover, the selectivity with less non-specific DNA adsorption was also expected and could be expected to be successfully applied for real samples detection demonstrating a potential application. In terms of the conceptual and fundamental mode of operation, three main compounds compose a biosensor including the bio-receptor, the transducer, and the amplifier. The sample analytes including antigen, protein or viral DNA, can be detected by bio-receptor (antibody, enzyme, DNA, aptamer, respectively) immobilized on the transducer, which transforms the effect of the recognition process into an observable and measurable signal. The variation of the signal is proportional to the concentration of the target in the solution and can be measured by different techniques such as optical 2 , thermal 3 , piezoelectric 4 and electrochemical 5 methods (Fig. 2).

Fig. 2. Schematic presentation of biosensors

Although numerous methods have been used for the detection and identification of biomolecules, these biosensors suffer from certain drawbacks. For instance, optical biosensors, though very sensitive, however, cannot be used in turbid media 6 . Thermal biosensors cannot be utilized with systems with very little heat change. Moreover, they are difficult to handle, necessary to use large equipment. Typically electrochemical method, due to the possibility of miniaturization, low cost, and direct electric signals 7 , has emerged as the most commonly used biosensors and becomes an attractive technique for identification of biomolecules. They have been found to overcome most of the disadvantages, which inhibit the use of other types of biosensors.

Electrochemical biosensors, due to their rapidement, easy to handle and low cost, become the most commonly used class of biosensors 8 . Depending upon the electrochemical property to be measured by a detector system, electrochemical biosensors may further be divided into conductometric, potentiometric and amperometric biosensors 4 . References are also made to other types of electrochemical detection techniques, such as impedimetric, which measures impedance (both resistance and capacitance), and field-effect, which uses transistor technology to measure current as a result of a potentiometric effect at a gate electrode 3 .

According to the nature of the biomolecules immobilized on the surface or the reactions, biosensors are classified in three categories immunosensors, enzyme sensors and DNA 22 sensors. A basic DNA sensor is designed by the immobilization of a single strand oligonucleotide (ssDNA) on a transducer surface to recognize its complementary single strand DNA (cDNA) sequence via hybridization. Due to the specific and robust interaction between complementary sequences, DNA sensor is especially suited for biosensor applications.

Therefore, combing electrochemical technology with nucleic acid layers, the new concept of the electrochemical DNA hybridization biosensor was first introduced by Millan and Mikkelsen back in 1993 9 , this exciting research area has received intense attention from several groups around the world, an impressive number of inventive designs for electrochemical DNA sensors have appeared and promised to provide a simple, accurate and inexpensive platform for patient diagnosis. 7 In my thesis, I will focus on electrochemical DNA sensor where the different technologies and principles will be described.

Performance of biosensor is characterized by some important parameters such as dynamic and linear range of detection, limit of detection (LOD), sensitivity, and selectivity (Fig. 3). Dynamic range represents concentration of analyte where a variation of the electrochemical signal is observed. The slope of the curve plotted in the linear range corresponds to the sensitivity of the biosensor. The lower value measured corresponds to the limit of quantification (LOD) and by extrapolation of this curve the detection limit (DL) could be calculated according to various methods described in analytical techniques. The most commonly used in biosensors devices concerns those taking into account signal to noise ratio, where DL is obtained by the equation:

Where sbl is the standard deviation, o is the measurement obtained with blank test and 1 is the sensitivity obtained by the slope of the linear part of the calibration curve.

Fig. 3. Presentation of parameters which characterize biosensors

The key issues in the development of all biosensors include design of the transducers so that the analyte selectively interacts with the sensing surface 10,11 for achievements of efficient transduction of the bio-recognition event 12,13,14 . For example, various materials such as nanoparticles, polymers, carbon materials or complex of both are developed as transducers of electrochemical biosensor. Meanwhile, in order to meet the higher requirement of DNA analysis in different fields, the choice of detection technologies are also important for increasing the sensitivity and selectivity of the biosensor [15][16][17][18] , and the response times in very sensitive systems 19 .

Thus, the detection technologies and the property of transducer have a significant impact on the performance of the biosensor.

DNA detection technologies

The pivotal detection of electrochemical DNA sensor is based on the hybridization reaction between ssDNA and complementary cDNA, which results in changes of the electric properties of the surface. Different transductions are developed and can be subdivided into two main categories, label-based and label-free approaches 20,21 .

Label-based electrochemical detection of DNA hybridization

DNA labelled with enzymes

Single strain DNA can be labelled by some enzymes such as horseradish peroxidase [START_REF] Wan | Ligase-based multiple DNA analysis by using an electrochemical sensor array[END_REF][START_REF] Zeng | Gold nanoparticles/water-soluble carbon nanotubes/aromatic diamine polymer composite films for highly sensitive detection of cellobiose dehydrogenase gene[END_REF] , alkaline phosphatase [START_REF] Carpini | Oligonucleotide-modified screen-printed gold electrodes for enzymeamplified sensing of nucleic acids[END_REF] and glucose oxidase [START_REF] Hajdukiewicz | An enzyme-amplified amperometric DNA hybridisation assay using DNA immobilised in a carboxymethylated dextran film anchored to a graphite surface[END_REF] . The basic principle is to label the DNA target with redox enzymes.

When the hybridization occurs with immobilized ssDNA probe on the surface, DNA labelled with enzyme remains on the electrode. Hybridization reaction could be followed through the catalytic properties of the enzymes in presence of their substrates where the obtained products are electroactive. In this case immobilized enzymes lead to production of electroactive species near the electrode surface, which gives a current related to the amount of hybridized DNA target (Fig. 4). For association of DNA with enzyme, some assays use affinity biotin/avidin system

DNA labelled with redox marker

The monitoring of hybridization between two complementary DNA strands could be performed by labelling of ssDNA probe with a redox marker, such as methylene blue (MB) 27- 29 or ferrocene (Fc) [START_REF] Anne | 3′-ferrocene-labeled oligonucleotide chains end-tethered to gold electrode surfaces: Novel model systems for exploring flexibility of short DNA using cyclic voltammetry[END_REF] . The electroactive molecules on the surface were detected directly by straightforward transduction. Ricci et al. [START_REF] Ricci | Surface chemistry effects on the performance of an electrochemical DNA sensor[END_REF] developed an electrochemical DNA sensor, with which the DNA sensing platform is based on DNA probe terminally labelled with redox marker MB. Because the DNA sensor performance is dependent on the nature of the selfassembled monolayer on the electrode surface, the signal changes reflected the dynamics of the probe before and after hybridization. (Fig. 6).

Fig.6. Schematic representation of DNA sensors based on MB as redox marker modified ssDNA probe

Moiroux et al. [START_REF] Moiroux | 3 ′ -Ferrocene-Labeled Oligonucleotide Chains End-Tethered to Gold Electrode Surfaces : Novel Model Systems for Exploring Flexibility of Short DNA Using Cyclic Voltammetry[END_REF] has developed a DNA sensor, where ssDNA oligonucleotides, labelled with a small redox marker, an alkylferrocene unit (Fc), was attached by the 5'-end to a gold electrode surface (Fig. 7). The authors follow the variation of current of Fc during the hybridization reaction. The disappearance of redox signal of Fc was observed after hybridization. Authors explained that the Fc, which was close to the surface (curve a), was sequestered after hybridization due to the formation of a rigid dsDNA chain that could not allowed it to be reduced and oxidized on the surface (curve b). After de-hybridization, the redox signal was recovered again, (curve c) or probe or changes in the electrochemical properties of the interface.

Direct detection based on redox properties of DNA bases

Observation of redox peaks of DNA bases, due to reduction and oxidation reactions, leads to monitoring of DNA hybridization. The most electroactive bases of DNA are guanine and adenine. Direct detection was reported for the first time by Palecek [START_REF] Paleček | Oscillographic Polarography of Highly Polymerized Deoxyribonucleic Acid[END_REF] , [START_REF] Paleček | Adsorptive transfer stripping voltammetry: Determination of nanogram quantities of DNA immobilized at the electrode surface[END_REF] . The author monitored DNA hybridization through reduction of DNA nucleotidic bases (Fig. 9). [START_REF] Bollo | Electrooxidation of DNA at glassy carbon electrodes modified with multiwall carbon nanotubes dispersed in chitosan[END_REF] . Additionally for signal amplification, the surface was modified with carbon nanotubes entrapped in chitosan. They obtained a linear relationship between amount of DNA and current corresponding to oxidation signal of guanine with 90.0 ppm dsDNA. However compulsory application of high potential for DNA oxidation is a drawback of this method.

Direct detection based on redox properties of redox markers in solution

In recent years, the sensitively label-free sensing technique of electrochemical impedance (EIS) is a useful tool for analyzing changes in interfacial properties of modified electrodes induced by binding of charged biomolecules on the surfaces. The changes in signal of different types of redox markers in solution near the electrode were detected by EIS.

According to the charge properties of transduces, the choice of the redox markers possessing different charges, negatively, neutrally or positively, has large effect on the performance of biosensors.

Cui et al [START_REF] Cui | A label-free electrochemical DNA biosensor for breast cancer marker BRCA1 based on self-assembled antifouling peptide monolayer[END_REF] sensor based on thionine-graphene (Th-G) nanocomposite modified gold electrode [START_REF] Zhu | DNA electrochemical biosensor based on thionine-graphene nanocomposite[END_REF] . An amino-substituted ssDNA probe was covalently grafted onto the surface of the thioninegraphene nanocomposite by the cross-linker glutaraldehyde (GA). The hybridization reaction on the modified electrode was monitored by differential pulse voltammetry analysis (DPV) using an electroactive daunomycin, in solution, as the redox indicator (Fig. 8). Under optimum conditions, the proposed biosensor achieved a wide range of 1.0x10 -12 to 1.0x10 -7 M with a good linearity (R2 = 0.9976) exhibiting high sensitivity and low detection limit of 1.26x10 -13 M (S/N = 3). In addition, the biosensor was highly selective to discriminate onebase or two-base mismatched sequences (Fig. 11).

Fig. 11. Schematic diagram of covalent immobilization of ssDNA probe on Th-G nanocomposite

Li et al. [START_REF] Li | An ultrasensitive and label-free electrochemical DNA biosensor for detection of DNase I activity[END_REF] s elaborated the working mechanism of label-free electrochemical DNA sensor to detect DNase I activity (Fig. 12) by monitoring the redox signal of the methylene blue (MB) which well known to bind the guanine bases of DNA via intercalation process [START_REF] Yan | Electrochemical DNA biosensor for the detection of specific gene related to Microcystis species[END_REF] Regarding literature different redox indicators or DNA-binding intercalators were used for the development of label-free DNA sensors which are resumed in the Table 1. [Co(phen)3] 3+ DPV 5.0×10 -12 to 1.0×10 -9 M 4.3×10 -13 M Liu et al. [START_REF] Liu | Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode[END_REF] Thus label-free monitoring of bio recognition events provides a promising platform, which is simple, cost-effective, and requires no external modification of the biomolecules.

The other essential role of the sensor is the choice of the transducer which provides a suitable platform that facilitates the immobilization of the DNA probe and provided a significant signal for electronic readout when hybridized DNA complex are formed. Recent researches are focus on the combination of different nanomaterials, with which endow additional properties as well as analytical performance of biosensors

Transducers for DNA sensor

Nowadays, in order to develop the higher requirement of DNA analysis, numerous nanomaterials have been employed significantly to construct electrochemical biosensors with the novel sensing platforms to improve the recognition property of the sensing layer and the signal output intensity of the sensing event for enhanced performance [START_REF] Xiao | Label-free electrochemical detection of DNA in blood serum via target-induced resolution of an electrode-bound DNA pseudoknot[END_REF][START_REF] Weng | Label-Free DNA Sensor by Boron-Doped Diamond Electrode Using an ac Impedimetric Approach[END_REF] . Some of them will be described in this document including polymers, nanoparticles or nanomaterials based on carbon materials. In each case, different and original approaches for immobilization of 32 ssDNA probes on the transducer surface have been employed such as adsorption [START_REF] Mohan | A label-free genosensor for BRCA1 related sequence based on impedance spectroscopy[END_REF] , selfassembly [START_REF] Gu | Electrochemical impedance sensing of DNA hybridization on conducting polymer film-modified diamond[END_REF][START_REF] Wang | Electrochemical detection of DNA immobilized on gold colloid particles modified self-assembled monolayer electrode with silver nanoparticle label[END_REF] , covalent binding [START_REF] Ozsoz | Electrochemical genosensor based on colloidal gold nanoparticles for the detection of Factor V Leiden mutation using disposable pencil graphite electrodes[END_REF][START_REF] Kerman | Modification of Escherichia coli single-stranded DNA binding protein with gold nanoparticles for electrochemical detection of DNA hybridization[END_REF] , biotin -avidin interactions [START_REF] Feng | A nano-porous CeO2/Chitosan composite film as the immobilization matrix for colorectal cancer DNA sequence-selective electrochemical biosensor[END_REF] and entrapment in a polymer matrix [START_REF] Tang | Preparation, structure, and electrochemical properties of reduced graphene sheet films[END_REF] .

Polymers

A class of organic polymers, known as conducting polymers (CPs), has become increasingly popular due to its unique electrical and optical properties. Several CPs are known to interact with biological samples while maintaining good biocompatibility and hence, CPs are applied for use in a numerous biological and medical applications [START_REF] Nambiar | Conductive polymer-based sensors for biomedical applications[END_REF] .

For example, Li et al. prepared a new type of conjugated CP, poly(indole-5-carboxylic acid) (PICA), on GCE by means of anodic oxidation (Fig. 13) [START_REF] Li | Label-free detection of DNA hybridization based on poly ( indole-5-carboxylic acid ) conducting polymer[END_REF] . PICA exhibited optimal electrochemical behavior and thermal stability, with a conductivity of 10 -2 S/cm and high redox activity compatible with the concept of molecular-wire transduction. The PICAmodified sensor showed comparable sensitivity and its detection limit was 1.0 nM, which can be further improved by increasing the side chain length because longer side chains permit greater freedom of movement and more enhanced hybridization [START_REF] Bangar | Single Conducting Polymer Nanowire Based Sequence-Specific , Base-Pair-Length Dependant Label-free DNA Sensor[END_REF] .

Fig. 13. Schematic representation of the preparation of an electrochemical DNA sensor based on a poly(indole-5-carboxylic acid) (PICA) conducting polymer

The summary of DNA sensors based on CP or CPC* were shown in Table 2.

Nanoparticles

Nanoparticles have numerous possible applications in biosensors. For example, functional nanoparticles bound to biological molecules (e.g. peptides, proteins, nucleic acids) have been developed for use in biosensors to detect and amplify various signals.

Electrochemical detection of DNA target immobilized on gold nanoparticles selfassembly modified electrode has been reported by Wang et al. (Fig. 14) [START_REF] Wang | Electrochemical detection of DNA immobilized on gold colloid particles modified self-assembled monolayer electrode with silver nanoparticle label[END_REF] Self-assembly of colloidal Au onto a cystamine modified gold electrode can enlarge the electrode surface area and enhance greatly the amount of immobilized ssDNA. The electron transfer process of [Fe(CN)6] 4-/ 3-on the gold surface was investigated by EIS and evaluated in the range of 10-800 pM with a detection level as low as 5 pM.

Fig. 14. Schematic illustration of the process of the immobilization of DNA onto the gold electrode

The electrochemical oxidation signal of Au nanoparticles anchored on a probe enabled the detection of factor V Leiden mutation in PCR samples [START_REF] Ozsoz | Electrochemical genosensor based on colloidal gold nanoparticles for the detection of Factor V Leiden mutation using disposable pencil graphite electrodes[END_REF] . Kerman et al. has reported that the unique binding event between Escherichia coli ssDNA binding protein (SSB) and singlestranded oligonucleotides conjugated to gold (Au) nanoparticles was utilized for the electrochemical detection of DNA hybridization (Fig. 15). 56

Association of nanoparticles and polymers

To further improve the performance of sensors combining various properties of materials, nanocomposites have attracted more attention in recent years.

Nanoparticles could embedded into porous over polymer film [START_REF] Miodek | E-DNA Sensor of Mycobacterium tuberculosis Based on Electrochemical Assembly of Nanomaterials (MWCNTs/PPy/PAMAM)[END_REF] that increase the electron transfer between the solution and the electrode, increase the immobilization surface of biomolecules on polymer. Feng et al. [START_REF] Feng | A nano-porous CeO2/Chitosan composite film as the immobilization matrix for colorectal cancer DNA sequence-selective electrochemical biosensor[END_REF] Nanoparticles of silver were combined with nanotubes of polypyrrole and poly(3,4ethylenedioxythiophene) (PPy-PEDOT) and have been investigated by Radhakrishnan 38 for the sensing of DNA hybridization using EIS as the detection method. The dynamic detection range was observed from 1×10 -11 to 1×10 -14 M with a detection as low as 5.4×10 -15 M (Fig. 16). (Table 3). PPy-MWCNT-AuNPs Avian influenza virus H5N1 4.3×10 -13 M Liu et al. [START_REF] Liu | Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode[END_REF] Among different nanomaterials, carbon nanomaterials hold potential promise as a material for designing a new generation of biosensors due to their unique characteristics.

Carbon materials

Carbon is an abundant element playing a major role in the chemistry life [START_REF] Lam | A review of carbon nanotube toxicity and assessment of potential occupational and environmental health risks[END_REF] . Various carbon allotropes exist including diamond (the carbon atoms are bonded together in a tetrahedral lattice arrangement, 3D), graphite (the carbon atoms are bonded together in sheets of a hexagonal lattice, 3D), graphene (2D), carbon nanotubes (single-walled carbon nanotubes (SWCNT) and multi-walled carbon nanotubes (MWCNT), 1D), and fullerenes (the carbon atoms are bonded together in spherical, tubular, or ellipsoidal formations, 0D) (Fig. 17). They all possess the capacity to hybridize into sp, sp 2 and sp 3 configurations with narrow gaps between their 2s and 2p electron shells and have very different physical and electrochemical properties such as wide potential windows, good electrocatalytic activity for many redox reactions [START_REF] Tang | Preparation, structure, and electrochemical properties of reduced graphene sheet films[END_REF][START_REF] Phiri | General overview of graphene: Production, properties and application in polymer composites[END_REF][START_REF] Atta | Graphene-A Platform for Sensor and Biosensor Applications[END_REF] . Fullerenes (C60) due to its unique and favorable characteristics of easy chemical modification, conductivity, and electrochemical properties, has tremendously used in biosensor applications. Hang et al. fabricated a novel DNA sensor based on polyhydroxylated fullerenol (FLN) as the probe DNA immobilization platform which could be stabilized with thioglycolic acid (TGA) via covalent interaction (Fig. 18). Due to 3D FLN spherical structure with plenty of grafting sites (-OH), the amount of probe DNA was greatly amplified and remained flexible. This promotes the sensing interface and the hybridization with the DNA target. Authors observed with a such DNA sensor a wide linear range from 1.0 fM to 1.0 nM with a detection limit down to 0.17 fM in target DNA detection. 64 DNA oligonucleotides can be immobilized onto the CNT-based electrode through physical absorption or ionic adsorption through a composite of polymer and CNT [START_REF] Li | DNA biosensor based on chitosan film doped with carbon nanotubes[END_REF] . However, covalent attachment plays more important role in CNT-based DNA electrochemical sensors as it lead to stable immobilization of DNA on CNT. Cai et al. [START_REF] Cai | Carbon nanotube-enhanced electrochemical DNA biosensor for DNA hybridization detection[END_REF] first reported the use of CNTs to fabricate an electrochemical DNA sensor for the specific DNA detection. A DNA probe with amino group at its 5'-phosphate end (NH2-ssDNA) was covalently bonded onto the CNTs-COOH-modified glassy carbon electrode (GCE) surface. CNT-modified electrode allows fast electron transfer between electrode and the redox intercalator daunomycin (Fig. 19). The detection limit achieved is 1.0 × 10 -10 M for complementary DNA. 20). This modified sensor then treated with avidin and biotinylated oligonucleotides related to the gene of N. gonorrhoeae with methylene blue (MB) as electroactive indicator. Using differential pulse voltammetry, the developed sensor showed high sensitivity with wide concentration range from 1×10 -19 M to 1×10 -6 M. Author reported that the aggregation of Fe3O4 nanoparticles due to its high surface area and magnetic dipole interaction between nanoparticles limite their application in biosensors field. They overcome this drawback by dispersing Fe3O4 nanoparticles in PANI-CNT composite and CNT worked as templates for the coating of Fe3O4 and PANI with minimum aggregation [START_REF] Singh | Nanobiocomposite platform based on polyaniline-iron oxide-carbon nanotubes for bacterial detection[END_REF] .

Fig. 20. A schematic diagram depicting the preparation of PANI-nFe3O4-CNT nanocomposite

Another example use MWCNTs to improve electron transfer ability in amperometric biosensors. Thus nanomaterial composed of MWCNTs coated with PPy acting as a transducer and redox dendrimers amplifying the electrochemical signal were reported a novel approach for highly sensitive amperometric detection of DNA. The biosensor was able of discrimination of DNA extracted from M. tuberculosis with single nucleotide polymorphism mutation which affair a drug resistance. [START_REF] Miodek | E-DNA Sensor of Mycobacterium tuberculosis Based on Electrochemical Assembly of Nanomaterials (MWCNTs/PPy/PAMAM)[END_REF] Improved electron transfer has been demonstrated compared to chemically modified surfaces, due to a homogeneous distribution of redox-active ferrocene bounded on the surface. The schematic represent of biosensor based on MWCNTs coated with PPy was shown in Fig. 21.

Fig.21. Schematic represent of biosensor based on MWCNTs coated with PPy

Despite many advantages and successful applications in biomedical engineering, there is a growing concern for safety with CNTs. Some recent studies have reported that the increased cytotoxicity of CNTs has been attributed to their agglomeration, induced oxidative stress, and incomplete removal of metal catalysts used to prepare CNTs [START_REF] Yang | Pharmacokinetics, metabolism and toxicity of carbon nanotubes for biomedical purposes[END_REF][START_REF] Lam | A review of carbon nanotube toxicity and assessment of potential occupational and environmental health risks[END_REF][START_REF] Firme | Toxicity issues in the application of carbon nanotubes to biological systems[END_REF] . In comparison with carbon nanotubes (CNTs), graphene is an ideal nanomaterial for various applications due to two obvious advantages superior to CNTs are shown as follows: 1. Graphene does not contain metallic impurities as CNTs do. In many cases, such impurities dominate the electrochemistry of CNTs and lead to misleading conclusions. 2. the production of graphene can be achieved using graphite that is cheap and accessible [START_REF] Pumera | Graphene for electrochemical sensing and biosensing[END_REF] . The properties of graphene are present in details in CHAPTER II.

42

CHAPTER Ⅱ: Graphene

Introduction-history

Graphene was discovered in 2004 by Geim and Novoselov [START_REF] Novoselov | Electric field effect in atomically thin carbon films[END_REF] and was isolated by mechanically cleaving of a graphite crystal. Graphene is a two-dimensional single layer of sp 2 hybridized carbon atoms arranged in a honeycomb lattice. It is also the base structure of all graphitic materials including graphite, carbon nanotubes and fullerenes (Fig. 22).

Fig. 22.

Basis of all graphitic forms. Reprinted form ref [START_REF] Chen | Progress in Materials Science[END_REF] Graphene has numerous electronic, mechanical, thermal, optical and chemical properties that make it a promising nanomaterial for use in energy-storage materials [START_REF] Chen | Preparation of graphene by a low-temperature thermal reduction at atmosphere pressure[END_REF] , drug delivery system [START_REF] Chen | Preparation of graphene by a low-temperature thermal reduction at atmosphere pressure[END_REF] , biosensors [START_REF] Kuila | Recent advances in graphene-based biosensors[END_REF] , polymer composites [START_REF] Phiri | General overview of graphene: Production, properties and application in polymer composites[END_REF] , liquid crystal devices [START_REF] Du | Ionic liquid-functionalized graphene as modifier for electrochemical and electrocatalytic improvement: Comparison of different carbon electrodes[END_REF] , super-capacitors [START_REF] Pumera | Graphene-based nanomaterials for energy storage[END_REF] , nanoelectronics [START_REF] Craciun | Tuneable electronic properties in graphene[END_REF] and others. The Table 4 below lists some main characteristics and properties of graphene compared to fullerene and carbon nanotubes one dimension [START_REF] Chen | Progress in Materials Science[END_REF] .

Graphene synthesis and its derivatives

A variety of methods have been developed to synthesize single or few-layer graphene sheets [START_REF] Lonkar | Recent advances in chemical modifications of graphene[END_REF] . It is recently known that graphene can be grown via several different methods, which can be divided into two categories including epithaxial technique and the exfoliation methods. For each synthesis, the challenge remains to achieve complete and homogeneous dispersion of individual graphene sheets in various solvents [START_REF] Kotov | Carbon sheet solutions Proteins downhill all the way[END_REF] . Indeed, graphene sheets tend to form irreversible agglomerates through Van der Waals interactions due to their conjugated and planar structure. So, the prevention of aggregation is essential for graphene sheets because most of their unique properties are only associated with individual sheets.

Epithaxial technique

According to the different application of substrates, epithaxial technique was classified into two groups: metallic substrate and non-metallic substrate. Fig. 23 illustrates typical epithaxial processes. The hydrocarbon molecules (called E) are deposited on the surface, undergo decomposition via a series of dehydrogenation reactions giving rise to various CxHy species shown as Ed, and H atoms. The new species are all able to diffuse across the surface. Smaller carbon species M and D form and diffuse on the surface, aggregating into larger clusters C. H atoms of the original molecule migrate on the surface, and form H2 molecules which evaporate from the surface. Finally, some of the species like M and D, or even their bigger clusters C, may attach to the island G at its edge.

Fig. 23. Schematic representation of typical epithaxial processes

Chemical vapor deposition (CVD) is a thermal method based on growth of graphene by dissolving carbon atoms onto a catalytic metallic substrate, such as Ni, Cu, Co, Pt, Ir and followed by precipitating out after cooling to form large-scale graphene films 82 . The carbon atoms comes from a carbon-containing gas such as methane, ethane, or propane, decomposed at high-temperature. For example, the schematic representation of CVD process on Ni substrate was shown in Fig. 24. This technique was used to fabricate multilayer graphene nanoflake films [START_REF] Nai | Catalyst-Free Efficient Growth , Orientation and Biosensing Properties of Multilayer Graphene Nanoflake Films with Sharp Edge Planes[END_REF][START_REF] Sutter | Epitaxial graphene on ruthenium[END_REF] , but the films are actually composed of 2-3 nm thick graphite nanoflakes rather than a single sheet.

Meanwhile, it requires additional transfer steps to fabricate the devices, since the CVD graphene is produced on certain metallic substrates, leading to contaminations and destruction of graphene sheets.

Fig. 24. Schematic representation of CVD processes on Ni substrate

Efforts are also made to grow graphene on silicon carbide (SiC) wafers via sublimation of silicon atoms 85 , which allows the production of a large area of single layer of graphene with a high yield up to 95%, a high conductivity and high-quality (Fig. 25A). The thermal decomposition of SiC appears the most promising from the perspective of electronic device fabrication, thanks to its direct-growth process on a semiconductor surface, and the extent of control on number of layers, quality, and uniformity obtained. However, bulk SiC substrates present limitations in terms of costs, sizes, and difficulty in micromachining.

Direct growth of graphene on hetero-epitaxial cubic silicon carbide (3C-SiC) on silicon (Si) substrate would overcome above-mentioned limitations [START_REF] Ouerghi | Epitaxial graphene on 3C-SiC (111) pseudosubstrate : Structural and electronic properties[END_REF][START_REF] Miyamoto | Regular of epitaxial graphene layers on 3C SiC/Si (111) as function of annealing temperature[END_REF] (Fig. 25B).

Two major advantages by associating with Si are first, 3C-SiC on Si substrate wafers are at the moment less expensive than 3C-SiC by orders of magnitude, large size up to 12 inches could be obtained and finally, the Si-based substrate provides easy access to integrated circuit technology and infrastructure. However, in the case of silicon carbide on silicon, the quality of the graphene film produced in high/ultra-high vacuum is limited due to the difficulty in controlling sublimation rates at relatively low temperature. In compared to the exfoliated graphene, a considerably higher ID/IG ratio about ~1 was indicated by Raman [START_REF] Gupta | Evolution of epitaxial graphene layers on 3C SiC / Si ( 1 1 1 ) as a function of annealing temperature in UHV[END_REF][START_REF] Suemitsu | Graphene Formation on a 3C-SiC(111) Thin Film Grown on Si(110) Substrate[END_REF] .

Fig. 25. (A) Growth of epitaxial graphene on silicon carbide wafer (B) 3C-SiC on Si substrate wafer via sublimation of silicon atoms

Exfoliation methods

Physical mechanical exfoliation

Mechanical exfoliation is a simple peeling process from a commercially highly oriented pyrolytic graphite (HOPG) sheet (Fig. 26A). This technique, also called "scotch-tape" method was first employed by Novoselov et al. [START_REF] Novoselov | Electric field effect in atomically thin carbon films[END_REF][START_REF] Dan | Intrinsic response of graphene vapor sensors[END_REF] . For fundamental research, this kind of graphene is incontrollable in shape, size and location. Hence the yield of devices is extremely low, and such pristine graphene is more suitable only for theoretical study and proof-of-concept demonstration. Graphite can also be exfoliated in presence of small molecules (N-methyl-pyrrolidone [START_REF] Blake | Graphene-Based Liquid Crystal Device[END_REF] or surfactant sodium dodecylbenzene sulfonate, SDBS) [START_REF] Lotya | Liquid Phase Production of Graphene by Exfoliation of Graphite in Surfactant / Water Solutions[END_REF] that intercalate between the layers of graphite to produce monolayer graphene. (Fig. 26B) However, the disadvantage of this process is the high cost of the solvent.

Electrochemical exfoliation of graphite

Electrochemical techniques are alternatively used to produce graphene flakes because they are simple, economic, non-destructive, environmentally friend, operates at ambient temperature and pressure, and the thickness which could be controlled by adjusting the electrode potential. In recent years, some researchers have attempted to produce graphene sheets by using electrochemical exfoliation of graphite anode, which was first reported by Lotya et al. in 2008 [START_REF] Lotya | Liquid Phase Production of Graphene by Exfoliation of Graphite in Surfactant / Water Solutions[END_REF] . Liu et al. [START_REF] Liu | One-Step Ionic-Liquid-Assisted Electrochemical Synthesis of Ionic-Liquid-Functionalized Graphene Sheets Directly from Graphite[END_REF] used a mixed solution containing ionic liquid 1-octyl-3-methyl-imidazolium hexafluorophosphate and water as electrolyte. Through intercalation of PF6-ions into the graphite anode assisted by oxidation of graphite edges by hydroxyl radicals, a dispersion containing graphene was obtained (Fig. 27). Alanyalıoğlu et al. controlled the thickness and order of graphene by using surfactant-assisted electrochemical processes at different intercalation potentials of 1.3, 1.4, 1.6, and 1.8 V vs. Pt [START_REF] Alanyalioglu | The synthesis of graphene sheets with controlled thickness and order using surfactant-assisted electrochemical processes[END_REF] .

Fig. 27. Schematic illustration of the graphene exfoliation process by electrohemical techniques

Chemically oxidized to GO and then reduced to RGO

The pristine graphite sheet is atomically flat with the Van der Waals thickness of ~0.34 nm. Graphene oxide sheets obtained from graphite are thicker due to the displacement of sp 3 hybridized carbon atoms slightly above and below the original graphene plane and presence of covalently bound oxygen atoms. This method is based on the oxidative exfoliation of natural graphite to graphene oxide (GO) followed by its reduction. Thus, this method is expected to obtain single layer. The reduction of GO sheets to RGO can be reduced via electrochemical, chemical or thermal reduction to restore the π-conjugated structure and conductivity. This is one of the most efficient methods for low-cost and large-scale production of single layer graphene 96 97,94 . In this case, Graphite oxide is usually synthesized through the oxidation of graphite in the presence of strong oxidants including concentrated sulfuric acid, nitric acid and potassium permanganate based on Hummers method [START_REF] Gupta | Evolution of epitaxial graphene layers on 3C SiC / Si ( 1 1 1 ) as a function of annealing temperature in UHV[END_REF] or modified Hummers' method [START_REF] Marcano | Improved synthesis of graphene oxide[END_REF][START_REF] Pham | One-step reduction of graphene oxide with l-glutathione[END_REF][START_REF] Sheng | High-performance self-assembled graphene hydrogels prepared by chemical reduction of graphene oxide[END_REF] . The graphite oxide is decorated by hydroxyl and epoxy functional groups on the hexagonal network of carbon atoms with carbonyl and carboxyl groups at the edges. Graphite oxide is highly hydrophilic and forms stable aqueous suspensions due to the large number of oxygen-containing functional groups as well as the repulsive electrostatic interactions at the edges of the platelets 100,101 .

Zhang et al. has reduced graphene oxide film electrochemically by cyclic voltammetry technique in 6 M KOH aqueous solution.

The ratio of oxygen/carbon content was significantly decreased after electrochemical reduction 102 . The drawback of this reduction is the intensive alkaline solution, which is adhesive and difficult to remove. More recently, many researches have been reported the conversion of graphene oxide (GO) into chemically reduced graphene oxide (CRGO) by chemical treatments 103,104,105 Chemical reduction of GO sheets has been performed with several reducing solvents including hydrazine 106,107,108 and sodium borohydrate 109,110 . The reestablishment of the conjugated graphene network could be attributed to the reaction pathway proposed by Stankovich et al 100,111 (Fig. 28) However, the carboxylic acid group were unlikely reduced by hydrazine. Another possible route GO was preformed using sodium borohydrate (NaBH4) in aqueous solution, where sodium borohydrate is more effective than hydrazine as a reductant although it can be hydrolyzed by water slowly. The C/O ratio was as high as 13.4:1 compared to 6.2:1 for hydrazine.

Fig. 28. (a) Oxidation of graphite to graphene oxide and reduction to reduced graphene oxide. (b) A proposed reaction pathway for epoxy reduction by hydrazine.

Thermal reduction is another approach to reduce GO to reduced graphene oxide.

It utilizes the heat treatment to remove the oxide functional groups from graphene oxide surfaces. Aksay's group have exfoliated and reduced stacked GO by heating GO to 1050 ℃, where oxide functional groups were eliminated as carbon dioxide 85,112 .

It was calculated that high pressure as high as 2.5 MPa was required to separate GO sheets by numerically evaluating the Hamaker constant while the pressure generated during the exfoliation was 1-2 orders of magnitude higher. Although the thermal reduction can produce 80% single layer RGO, the left vacancies and structural defects may affect the electrical and mechanical properties of RGO. However, the conductivity was measured to be 1000-2300 S/m, suggesting the effective reduction and restoration of electronic structures from GO.

Recently, Dubin et al. elaborated a simple one-step solvothermal reduction method to produce reduced graphene oxide by thermal deoxygenation of GO at 200 ℃ refluxing in N-methyl-2-pyrrolidinone (NMP) as reductant agent. The RGO layers remained in a stable dispersion after the reaction. This approach provides a simple, low-temperature method to produce reduced graphene oxide, which is considered as an excellent way to synthesize RGO, in spite of structural defects inevitably as indicated by Raman spectroscopic studies 104 .

One of the challenge in the development of graphene-based sensor devices is that graphene or reduced graphene oxide (RGO) are hydrophobic and tend to agglomerate in water, and even restack to form graphite owing to van-der Waals interactions and strong π-π stacking 113,114 . As a result, considerable effort has been expended in improving the compatibility and dispersion of graphene. Recently, a vast majority of researches on the enhancement of dispersion and self-assembly properties of RGO was focus on the modification and entrapment of functional moieties tuning graphene's dispersion and performance to achieve the best performance of DNA sensors, which will be elaborated in next section. In this respect, a majority of work to make RGO a viable option for applications through both covalent interactions and noncovalent bonds. In contrast, the noncovalent functionalization is largely preferred as it does not alter the π-conjugated structure and the electronic properties of graphene, 51 while it simultaneously introduces new chemical groups on the surface 115 . In addition, the mild and nonpermanent character of noncovalent interactions offers high flexibility with regards to the treatment of the afforded products.

Electrochemical DNA sensors based on graphene

The graphene surface has been selected as the excellent substrate for DNA detection because its surface and structure is independent of pH, salt concentration and so on. 116 . Different approaches are described in the literature, to attach singlestranded DNA strand (ssDNA), on the graphene. These techniques include direct adsorption of DNA onto graphene through weak interactions, covalent attachment by different functional groups (amide, ester) or by using materials (nanoparticules, polymers) or aromatics compounds. The way to fix DNA onto the surface is crucial in the construction of sensor, in term of orientation, stability and accessibility.

Through materials like nanoparticles and polymers

The graphene-nanoparticle hybrid structures are particularly interesting because 53 not only do they display the individual properties of the nanoparticles and of graphene, but they can also exhibit additional synergistic properties thereby enhancing the achievable sensitivity and selectivity using a variety of sensing mechanisms.

Through aromatics compounds

Aromatic molecules have large aromatic plane and can anchor onto the reduced graphene oxide nanosheets surface without disturbing its electronic conjugation, providing stability for reduced graphene oxide. The most popular are perylene tetracarboxylic acid 120 and pyrenebutyric acid 121 (Fig. 31).

Fig 31. The structure of the aromatic molecules

The aromatic molecules act as wedges and penetrate slowly between the planar graphene layers usually by the assistant of sonication. Meanwhile, the aromatic molecules functionalization with RGO lead to a variety of terminated groups, which can not only remarkably improve the dispersibility and further functionalization and modification, but also offer opportunities for sensing performance.

For each approach of immobilization, some examples of DNA sensor based on RGO are presented as followed.

DNA sensors based on RGO with nanoparticles

The graphene-nanoparticle hybrid structures as well as their application in electronic, electrochemical sensors will be shown in detail.

Silver nanoparticles (AgNPs), as a typical noble metal nanoparticles, are extensively used in DNA hybridization sensing for amplification of hybridization event owing to their catalytic properties and also exhibit the high electrical and thermal conductivities 122 . The scheme was shown in Fig. 32. Extensive research has been carried out on RGO-based non-covalent functionalized electrochemical biosensors, the development of highly sensitivity and selectivity using self-assembled nanomaterial platform was imperative.

DNA sensors based on RGO with conductive polymers (CPs)

Fabrication of electrochemical sensors based on RGO-CPs becomes a popular approach in many electroanalytical investigations and shows good potential for constructing the sensors with high sensitivity and selectivity to detect target molecules based on different analytical strategies.

Various polymers, such as poly(methyl-meta acrylate) (PMMA), 123,124 poly(dimethylsiloxan) (PDMS), 125 polypropylene (PP) 126 are used in biosensing and mirco-array technologies.

DNA sensors based on RGO with aromatic molecules

Lee et al. 127 used a tetrapyrene derivative functionalized by an oligoetherdendron to exfoliate graphite and stabilize graphenic layers. The tetrapyrene aromatic rings interacted with RGO, while oligoether chains induce a high hydrophilic character to the nanomaterial. The authors elucidated that the same tetrapyrene derivative is unable to disperse SWNTs, indicating that the planarity of the carbon nanostructure is a crucial factor for the formation of an effective π stacking.

Bose et al. 121 employed the organic 9-anthracene carboxylic acid (9-ACA) to 55 improve the dispersibility in aqueous solution, which penetrates deeper inside the graphite flakes and finally exfoliates to form individual layers. The out-of-plane -COOH group helps in achieving stable dispersion in water and strong π-π interaction prevents the exfoliated sheets from restacking. The proposed mechanism for the formation of stable aqueous dispersion was shown in Fig. 33. 3,4,9,10-Perylene tetracarboxylic acid (PTCA), bearing π-conjugated perylene ring and carboxylic acid groups, was used to decorate graphene (Fig. 34). Based on the π-π stacking and hydrophobic interactions between the perylene ring and the basal plane of graphene, PTCA molecules separated graphene sheets efficiently and decorated them with plenty of -COOH. This method resolved the graphene aggregation problem, and the resulting hybrids dispersed well in aqueous solution.

After activation, NH2-ssDNA was covalently immobilized on the hybrid, which presented as lying structure. Hybridization to cDNA induced the lying ssDNA to standing dsDNA, which caused the graphene interfacial property changes, such as negative charge and conformation. The porphyrins are aromatic tetrapyrrolic macrocycles with 22 π electrons, 18 of which are involved in aromaticity. Three main types of carbon atoms can be distinguished in these molecules, which are noted respectively α, β and meso. We have depicted (Fig. 35) the simplest porphyrins (called porphine) and the carbon atoms were numbered according to the IUPAC nomenclature.

Fig. 35. Structure of porphine 58

The porphyrins can be classified according to the substituents carried by the β or meso carbon atoms and by the nature of the metal complexed by the macrocycle. The term free base is used in the absence of metal and metalloporphyrin in the opposite case. We show below two conventional examples of meso-substituted porphyrins free (tetraphenylporphyrin) and metaled with zinc.(Fig. 36)

Fig. 36. two examples of structure of free and metaled porphyrin

Optical and electrochemical Properties

Because of their particular electronic structure, porphyrins are highly colored molecules. They present two types of absorption bands in the ultraviolet and visible domains, attributed to transitions π-π *:

a very intense band (ε> 10 5 mol -1 .L.cm -1 ) located between 380 and 450 nm, called band Soret. It is characteristic of the aromaticity of the molecule.

-a series of bands in the visible called Q bands, less intense (ε ~ 10 4 mol -1 .L.cm -1 ) but nevertheless responsible for the color of the porphyrins. The number of Q bands generally makes it possible to distinguish the free-base porphyrins and the metalloporphyrins. The free bases have four bands Q whereas the metalloporphyrins, which have a larger symmetry, have only two (or even one) in general. The energy and intensity of these bands depend on the substituents of the porphyrin, the metal, the additional ligands on the metal, the macrocycle deformations, the solvent and the interactions between chromophores in the multi-porphyrinic buildings.

2. Synthesis of meso substituted porphyrin

These last fifteen years, the synthesis chemistry of meso-substituted porphyrin has considerably been developed and improved, decreasing the purifications step, the number of regioisomers synthesized 148 . Today, there are three ways to access such molecules (scheme. 37). The first approach consists in a mixed condensation using pyrrole and various aldehydes 149 (path A). In this case, the number of regioisomers Differentially functionalized porphyrins can also be prepared by using two different aldehydes under essentially the same conditions. The Adler-Longo method is often used to obtain unsymmetrically substituted tetraphenylporphyrins with groups suitable for further modification.

While the above methods show the utility of the propionic acid method, there are several drawbacks. The formation of the reduced porphyrin (chlorin) invariably contaminates the product and a high percentage of by-products are also formed.

Another problem is the failure of the reaction with aldehydes containing acid sensitive functional groups. These drawbacks associated with the rather harsh conditions could be overcome using method developed by Lindsey et al 154 . The Lindsey method relies on the existence of the formation of porphyrinogen as an intermediate in porphyrin synthesis. After the equilibrium has been established, an oxidant is added which converts the porphyrinogen to the corresponding porphyrin irreversibly (Fig. 38). The advantage of this method is that it allows the formation of porphyrins from acid sensitive aldehydes, in higher yields, with more facile purification. 39

. The method involves use of one dipyrromethane bearing two formyl groups αpyrrolic nitrogen and another dipyrromethane with no α-substitution.

Fig. 39. 2+2 Porphyrin synthesis by MacDonald

There is obvious scope for the preparation of a large number of functionalized porphyrins using [2+2] route, and a great of regioselectivity can be attained compared to the Adler and Lindsey methods. The 2+2 route has become very popular in recent years due to its flexibility, and there are many reports of porphyrin synthesis based on this method. It is, however, very dependent on the availability of suitable dipyrromethane building blocks, and the preparation of dipyrromethanes has also been the subject of several recent studies.

Interaction between porphyrins and graphene

The great similarity of structure (planarity, conjugated network..) and the ability for both to introduce different substituants on the aromatic system, as previously showed, promote the association of graphene and poprhyrins.

For example, the presence of carboxylic groups on the edge of RGO structure allowed the covalent attachment of the protoporphyrin IX on the sheets of RGO in presence of coupling agent 156 . The porphyrin molecules were thereby covalently attached to the surface of RGO via a benzene ring. The preparations are shown in Fig. 40.

Fig. 40. Preparation of RGO-TPP using diazonium reaction

Authors demonstrated that attachment of the porphyrin units to the surface of the RGO by diazotization significantly improves the solubility and ease of processing of these RGO-based nanocomposites.

Another approach consisted to use some nanoparticles of gold previously deposited on RGO to fix a sulfhydryl-porphyrin through S-Au bond 160 (Fig. 41).

Fig. 41. The preparation process of porphyrin/AuNPs/graphene nanocomposites

Finally, the most common approach is to associate porphyrins and graphene through non-covalent interactions including  stacking interactions. In this case, this mode of associations don't disturb the intrinsic physical and chemical properties of graphene. The introduction of charged on porphyrins can promote the dispersion of graphene in aqueous solution and avoid thus its agglomeration. Thus, a meso-tetra(4carboxyphenyl) cobalt porphyrin (CoTCPP) 161 can be strongly adsorbed on graphene through π-π stacking and hydrophobic interactions to avoid graphene aggregation and introduce more negatively charged -COOH groups and functionalities on graphene 63 surface, without destroying the conjugated π-system of graphene. Another example of charged tetraphenylporphyrines 133 , a positively (TPP-ammonium) and a negatively (TPP-SO3Na) molecules have also been used as dispersant for preparation of chemical reduced graphene oxide nanohybrides (Fig. 42).

Fig. 42. Molecular structures of CO-TCPP, TPP-SO3Na and TPP-ammonium

They have found that the π-π interactions taking place between porphyrins and RGO, as well as the porphyrin molecular architecture, play an important role in producing highly dispersible RGO sheets in water. Somes applications for employing porphyrin-graphene nanocomposites.

Oxygen Reduction Reaction

Jahan et al. 162 The detection limit was 4.6 nM for HQ (Fig. 44). In this case, the porphyrin exhibits its catalytic properties for the oxidation of hydroquinone into quinone. The hybrid nanomaterials which combined the features of both graphene (high conductivity and large specific surface area) and porphyrins (physical and chemical properties and electron transfer ability) were successfully synthesized. To investigate the characteristic properties of asprepared nanomaterials, scanning electron microscopy (SEM), atomic force microscopy (AFM), UV-visible spectra, Fourier Transform infrared spectra (FT-IR), X-ray photoelectron spectra (XPS)

and Raman spectroscopy were utilized. We confirm that the porphyrins interacted with CRGO and enhance the dispersion of graphene nano-sheet. The formation of hybrid nanomaterials through non-covalent interaction have no effect on electronic properties of chemically reduced graphene oxide (CRGO) and simultaneously introduce effectively functionalized groups on the surface of CRGO. Observed by AFM, the H2TPP-4CP with more negative charge is anchored at the planelike edges of CRGO which is considered as the electrochemical active centers through π-π stacking and hydrogen bond favoring the electron transfer process through hybrid nanomaterials.

The electrochemical properties was explored by electrochemical impedance spectroscopy (EIS)

and cyclic voltammetry (CV), which both confirm that the tight interaction exists between H2TPP

and CRGO, and the hybrid nanomaterial H2TPP-4CP/CRGO possesses more negative charge due to the carboxylic groups on the surface introduced by attached porphyrins. The superior conductivity of the hybrid nanomaterial is proven through charge transfer resistance measured by EIS. Also, the value of n which reflects the charge distribution of the surface decreases from 1 to 0.5 obtained by the fitting data of EIS illustrating the increase of plane-like edge after modification process. The electrochemical process and reversibility monitored in redox marker pbenzoquinone/hydroquinone, the neutral redox marker without charge ligands demonstrates that the hybrid nanomaterials possess high electroactivity with enhanced ks calculated by Nicolson model, compared to CRGO. The incorporation of manganese (III) is favor to further investigate properties of the nanomaterial through its electrochemical signal variation of the Mn 3+ /Mn 2+ redox complex. The ability of CRGO facilitates to immobilize a large amount of porphyrin. The advantages of such hybrid nanomaterial explore a high scope to their application in electrochemical biosensor.

Materials and Methods

Reagents

Graphene

Instruments and measurements

1 H and 13 C NMR spectra were recorded on Bruker AC 250 and Bruker AV 360 spectrometers (63 and 90 MHz for 13 C, respectively). Chemical shifts , are reported in ppm taking residual CDCl3 as the internal reference.

Mass spectra were recorded on a Finningan-MAT-95-S, using MeOH/CH2Cl2/H2O (45/40/15, v/v) as solvent.

Scanning Electron Microscopy (SEM) images were acquired using a ZEISS SUPRA TM 55VP GEMINI®.

Atomic Force Microscopy. A commercial dimension 3100 (Vecco Instruments, USA) atomic force microscope was used for topographical characterization of the samples. All measurements were performed in the tapping mode using rectangular silicon AFM tip.

UV-visible spectra were recorded with uvikon xl spectrometer.

Fourier Transform infrared spectra (FT-IR) were measured using a Bruker IFS66 FT-IR spectrometer equipped with a MCT detector and an attenuated total reflectance (ATR) crystal of germanium.

X-ray photoelectron spectroscopy (XPS) were conducted with a VG ESCALAB MKII spectrometer. The XPSPEAK software (Version 4.1) was used to deconvolute the narrow-scan XPS spectra of the C1s of the samples, using adventitious carbon to calibrate the C1s binding energy (284.5 eV).

Electrochemical measurements were performed using an AUTOLAB PGSTAT 100 electrochemical analysis system controlled by NOVA software (Methrom). The three-electrode cell was purchased from BASI and consisted of glassy carbon electrode (GCE) (surface 0.071 cm 2 ) as working electrode, a platinum as counterelectrode and Ag/AgCl as reference electrode. The analysis were performed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV).

EIS analysis were performed in [Fe(CN)6] 3-/4-at 0.15 V vs. Ag/AgCl at DC potential of 10 mV with the frequency ranging from 100 KHz to 0.1 Hz. The impedance measurements in PBS solution were obtained at -0.3 V vs. Ag/AgCl at DC potential of 10 mV with the frequency ranging from 100 KHz to 0.01 Hz.

CV analysis were realized in 0.1 M KCl with 5 mM [Fe(CN)6] 3-/4-species in the range of potential from -0.2 to 0.6 V at various scan rates in the range 0.005-1 V s -1 and also analyzed in 0.1 M KCl with 1 mM benzoquinone in the range of potential from -0.4 to 0.2 V at various scan rates in the range 0.05-1 V s -1 .

3.

Synthesis and preparation of nanomaterials The synthesis of porphyrins is known to be tedious, long and complex 1 . Herein, we propose, a simple and effective way to prepare the tetraphenylporphyrin bearing one carboxylic group (H2TPP-1CP) from the 5-(p-methoxyphenyl)triphenylporphyrin in three steps (Scheme 1.1). 5-(p-methoxyphenyl)triphenylporphyrin was first obtained in one step via a [2+2] condensation reaction from dipyrromethane and benzaldehyde with a good yield (20%). 2 The synthesis process is shown in scheme 1.1. After deprotection in acidic conditions, porphyrin bearing the hydroxy group was then condensed with 5-bromopentanenitrile by Willamson's condition to afford compound modified with nitrile. To obtain carboxylic porphyrin (H2TPP-1CP), nitrile was hydrolyzed by 12M HCl and afforded the modified porphyrin with carboxylic group with good yields 32% (Scheme 1.2). The experimental section is shown in details in Supporting Informations.

Synthesis of porphyrin functionalized with four carboxylic groups

The symmetrical tetraphenylporphyrin bearing four carboxylic groups (H2TPP-

4CP, 9) is prepared from the 5, 10 , 15, 20-tetrakis (p
methoxyphenyl)triphenylporphyrin ( 6) in three steps (Scheme 1.4) following the same procedure as H2TPP-1CP. Due to the symmetry of the porphyrin (6), the strategy applied for the synthesis is Adler and Longo, in one step via a condensation reaction from four equivalents of pyrrole units and four equivalent of p-methoxybenzaldehyde in propionic acid (Scheme 1.5). After simple purification, porphyrin ( 6) is obtained with a good yield (30%).

H N N N H N H 3 CO OCH 3 OCH 3 H 3 CO i H N N N H N HO HO OH OH ii H N N N H N RO RO OR OR R = -(CH 2 ) 4 -CN 8 R = -(CH 2 ) 4 -

Synthesis of graphene via electrochemical exfoliation of graphite (EEG)

The electrochemical reduction of graphite rods was performed following the procedure The reduction of GO by electrochemical methods to obtain ERGO was carried out following the method described by Zhang et al 4 . It was performed after the immobilization of GO on the surface of GCE. The modified electrode was introduced in a 6 M NaOH and the reduction was performed by scanning the potential from -1.6 to 0 V vs. Ag/AgCl for 8 cycles at 50 mV.s -1 . The modified electrode was then washed with distilled water.

Synthesis of RGO by using GO via chemical methods (CRGO)

The commercial GO was firstly dispersed in DMF (0.5 mg mL -1 ) under ultrasonically treatment for 30 min, and then heated in an oil bath (153 º C) for 1 h to obtain CRGO. As seen in Fig. 1.2, the color of the dispersion changed from brown to black, initially signaling the conversion of GO into CRGO. Afterwards, CRGO was obtained after centrifugation under the rate of 10000 rpm and separated from the DMF solution. Successive centrifugations in water and in ethanol were done to wash CRGO sheets and then dried in oven overnight 5 .

Fig. 1.2.

The photograph of GO in DMF before (left) and after (right) reduction. GO is generally highly oxygenated, bearing hydroxyl, epoxide, diol, ketone and carboxyl functional groups that disturb the van der waals interactions and significantly improve solubility in water and solvents. Chemical reduction of GO removes the oxygen functional group from the surface. The reduction of GO also favors the formation of hetero-structure with plane-like edge and nano band which is electrochemical active compared to basal plane electrochemically inert 6 .

Thus depending on the methods of reduction the electrochemical properties and the rate of homogenous electron transfer should be improved.

To intuitively evaluate the reduction level and determine the composition of the obtained CRGO sheets, X-ray photoelectron spectroscopy (XPS) was utilized. As shown in Fig. 1234, the C/O ratio in the exfoliated commercial GO increases remarkably after chemical reduction. The specific peaks at between 286 and 289 eV, which are assigned to epoxide, hydroxyl, and carboxyl groups remarkably decrease, revealing that the oxygen-containing groups were eliminated. Meanwhile, the intensity of the peak located at a binding energy of 284 eV, which is corresponding to sp 2 carbon, is much enhanced compared to that of peak centered at 285.5 eV (sp 3 carbon) 7 , demonstrating that the obtained CRGO has a high reductive level. The ratio of carbon content in GO and CRGO is estimated to be 45% and 86% respectively. In the same way the ratio of oxygen content decreases from 55 % for GO to 14% for CRGO (Table 1.1).

The result of XPS shown in Fig. 4D appears a new peak at 290.1 eV corresponding to the π-π* conjugation, indicating that the CRGO is compact with multi-layer stacks. Both these results are in agreement with Raman results and confirm that chemical reduction of graphene oxide leads to increase of conductivity due to decrease of oxide on the surface and increase of graphene sheet layers. For quasi-reversible electrode, of which peak potential separation is larger than 59.5/n mV, where n is the number of electron transferred of benzoquinone, although the cathodic peak current equals to its corresponding anodic one, the Nicholson method was used to calculate the rate constant 10 . Following the Nicholson's working curve, the ΔEp can be converted into a dimensionless kinetic parameter ψ for the coefficient of electron transfer α = 0.5, which is directly proportional to the reciprocal of the square root of scan rate v -1/2 (Fig. S1-3). The rate constant can thus be calculated by equation ( 1):

(

) 1
where ks is given in cm s -1 , the slope is obtained from a linear fit of ψ~ v -1/2 relationship, n is the number of electron transfer (n=2), F is the Faraday constant and D the diffusion coefficient of the redox marker benzo/hydroquinone in aqueous media which is obtained by the Randles-Sevcik equation 11 , ip = 2.99×10 5 nACD 1/2 v 1/2 (D= 1.17×10 -6 cm 2 s -1 ). The ks value is calculated to be 0.011 cm s -1 for CRGO, which is close to that of carbon pyrolysed at 800 ℃ (0.018 cm s -1 ) in the same order of magnitude 10 .

Interaction between porphyrins and CRGO 2.1 The association and dispersion

The hybrid nanomaterial suspensions were prepared by adding H2TPP into 1 mg mL -1 as-obtained CRGO, followed by ultrasonication for 30 min, the as-prepared H2TPP-1CP and H2TPP-4CP suspensions were kept in the fridge over one week. As observed in Fig. 1.7, the CRGO is aggregated and the precipitate is observed, in 90 contrast, the addition of porphyrin into CRGO suspension results in the formation of more stable and homogeneous hybrid nanomaterial suspensions, thus illustrates that the porphyrin functionalized with carboxylic group improves the solubility and dispersion of CRGO.

Fig. 1.8. SEM images of (a) CRGO and, (b) H2TPP-1CP/CRGO, and (c). H2TPP-4CP/CRGO

The surface topologies of CRGO and hybrid nanomaterials H2TPP-1CP/CRGO and H2TPP-4CP/CRGO have been also studied by atomic force microscopic (AFM).

Fig. 1.9a displays an image of CRGO sheets exhibiting flat surface and compact structure with roughness of 6nm. which is greater than the theoretical value for graphene nanosheets of 0.34 nm owing to the multi-layer of graphene sheets on the surface 12,13 and π-conjugated compact surface of CRGO. In the case of hybrid nanomaterials H2TPPs/CRGO, as we can see in Fig. 1.9b and c, nanosheets of graphene were formed, due to the association of porphyrins, which lead to the dispersion of CRGO. The roughness factor of hybrid nanomaterial H2TPP-1CP/CRGO is up to 250 nm, also due to the formation of small nano-sheet domains 92 and edge plane which are upward. From the result of Fig. 1.9c, it is noteworthy that the comparison of the images at the edges of the CRGO sheets is higher than that of interior regions (black circle in Fig. 1.9c). This phenomenon is likely caused by the presence of a higher density of functional groups where the association of porphyrins could be favored at the edge as observed also previously in the case of association of modified porphyrin with CRGO 14 .

Fig. 1.9. AFM images of (a) CRGO and, (b) H2TPP-1CP/CRGO, and (c). H2TPP-4CP/CRGO

Molecular characteristics and electronic properties of nanomaterials

The comprehensive exploration including the molecular characteristics and electronic properties of the interaction between H2TPP with different carboxylic groups and CRGO were performed through spectroscopic studies.

Molecular characteristics

Association between H2TPP and CRGO of hybrid nanomaterials was evaluated by UV-visible and FT-IR spectroscopies. The UV-visible absorption of complex H2TPP-1CP/CRGO (Fig. 1.10A) and H2TPP-4CP/CRGO (Fig. 1.10B) in DMF exhibits an intense Soret band at 417 nm characteristics of the electronic transition of complex from deeper π levels to LUMO and the four characteristic Q bands between

PART B: Results and discussion

Chapter I: Synthesis and properties of nanomaterials 93 500 and 700 nm corresponding to ππ* electronic transition from the HOMO to the LUMO 15 . After the association of CRGO there is no shift on the Soret band of porphyrin, indicating that the association of porphyrin and CRGO has no effect on the electronic properties of the macrocycle of porphyrin. However, a decrease in the intensity of the absorbance has been observed with the successive addition of CRGO with constant concentration of porphyrin, which confirmed the intimate interaction between H2TPP and CRGO. The spectra in Fig. 1.10A and B were further analyzed using the Benesi-Hildebrand expression by equation ( 2):

(

) 2
where Ka denotes the apparent association constant, ε is the molar absorption coefficient of porphyrin adsorbed on CRGO. Inverse of the difference absorbance at 417 nm (1/D417) was plotted versus inverse of the CRGO concentration (1/CCRGO).

Using the slope and the intercept of the linear fit we obtained the apparent association constant of H2TPP-1CP/CRGO and H2TPP-4CP/CRGO calculated to be 85.1 g -1 L and 64.4 g -1 L, respectively, which exhibit in the same order of magnitude of previous 56.6 g -1 . 16 However, the large apparent value of Ka shows that the interaction between H2TPP and CRGO sheets is rather strong and that it can be achieved in relatively dilute solutions. The as-prepared hybrid nanomaterial suspensions were dropped casting on GCE plates. The obtained samples were characterized by FT-IR spectroscopy. For H2TPP-1CP/CRGO, the FT-IR spectra (Fig. 1.11A) exhibits a band at 3256 cm -1 which is assigned to OH vibrations of carboxylic group of modified porphyrin and two stretching vibration bands in the double band region, at 1727 cm -1 corresponding to the C=O vibration and at 1577 cm -1 that can be attributed to C=C vibration. In comparison with H2TPP-1CP, the value of C=C shifts from 1611 to 1577 (33 cm -1 shift) due to π-π interaction between phenyl substituted porphyrin and π-conjugated CRGO. For H2TPP-4CP/CRGO (Fig. 1.11B), the analogous result is observed, 95 however, the red shift is merely 15 cm -1 , which is much lower than that of H2TPP-1CP/CRGO. The phenomenon is attributed to the less π-conjugation, probably due to the anchoring of H2TPP-4CP is more on the edges of CRGO instead of the basal plane.

The 21 cm -1 red shift of C=O value due to the formation of hydrogen bond between H2TPP-4CP and CRGO, however, scarcely shift for H2TPP-1CP/CRGO further demonstrate that the H2TPP-4CP is anchored at the edge of CRGO which is expected to enhance the electron transfer. The data of functional groups in details are shown in Transmitance (%)

Wavenumber (cm -1 ) CRGO 3248cm -1 1587cm -1 1583cm -1 C-H 2873cm -1 2959cm -1 1724cm -1 (B)

Fig. 1.11. (A) FT-IR spectroscopy and (B) FT-IR of (a) CRGO, (b) H2TPP-4CP, and (c) H2TPP-4CP/CRGO

The interaction between the CRGO and H2TPP were also characterized by using Raman spectroscopy (Fig. 1.12). The Raman spectra of CRGO show two broad peaks at 1352 and 1602 cm -1 , which correspond to the D-and G-bands of CRGO. After interacted with H2TPP-1CP and H2TPP-4CP, the intensity ratio of D/G was increased compared to that observed in CRGO. The increase of the ID/IG intensity ratio suggests that the average size of the sp 2 domain is decreased and the sp 3 domain is increased upon association of the H2TPP and also the formation of various graphene domains.

The formation of the various graphene domains could be related to the high dispersion process as observed previously in SEM and AFM images. The low shift of the G-band could be contributed to electron doping as a consequence of π-π interaction between porphyrin and CRGO 17,18 , which demonstrates that the porphyrin has little effect on the electronic properties of CRGO. The higher ID/IG ratio of H2TPP-4CP/CRGO, compared to the H2TPP-1CP/CRGO, confirming more defect of H2TPP-4CP/CRGO, which is coincide to the result of FT-IR. (3); where ε0 is the vacuum dielectric constant, ε1 is the double layer dielectric constant, A is the surface area and d is the thickness of the layer. The thickness of the H2TPP layers could be calculated from the capacitance value obtained from the second film of H2TPP layers. The value calculated to be 72 9.3 nm and 20.4 1.2 nm, respectively for H2TPP-1CP/CRGO and H2TPP-4CP/CRGO, further confirming that for H2TPP-1CP/CRGO, the nano-sheet is formed and the higher value, compared to H2TPP-4CP/CRGO, exhibits some aggregated domains upwards, and for H2TPP-4CP/CRGO, the porphyrin is anchored at the edge of CRGO, which is in agreement with the results of AFM. 1.15). The diffusion coefficient value was calculated to be 6×10 -6 cm 2 s -1 and 2.4×10 -6 cm 2 s -1 , respectively for H2TPP-1CP/CRGO and H2TPP-4CP/CRGO. Meanwhile, the calculated electron transfer constant ks of redox marker with negative ligants is affected by hybrid nanomaterials with negative charge. In the case of CV with negative redox marker, with the increase of carboxylic group, the reversibility becomes worse, which can be attributed to the strong electrostatic repulsion blocking the diffusion of negative ions to the electrode surface.

Fig. 1.15. Cyclic voltamograms for glassy carbon electrode covered by (A) H2TPP-1CP/CRGO, (B) H2TPP-4CP/CRGO obtained in 0.1 M KCl with 5 mM [Fe(CN)6]

3-/4-species in the range of potential from -0.2 to 0.6 V at various scan rates in the range 0.005-1 V s, and (C) shows the variation of anodic and cathodic current peaks vs. v 1/2 When the neutral redox marker p-benzoquinone/hydroquinone was employed for electrochemical characterization, the remarkably higher electrochemical process and high reversibility is observed for both hybrid nanomaterials (Fig. 1.16). The rates of 101 homogenous electron transfer (ks) followed by Nickolson model (Fig. S1.3) for H2TPP-1CP/CRGO and H2TPP-4CP/CRGO are calculated to be 0.028cm s -1 and 0.01 cm s -1 respectively, which is the same magnitude order of the value 0.01cm s -1 for CRGO and to a great extent increase the electron transfer of benzoquinone compared to bare GCE (0.0013 cm s -1 ) 10 , further elucidating that the addition of H2TPP-1CP/CRGO enhance the electron transfer and the addition of H2TPP-4CP/CRGO has no effect on the rate of electron transfer. The kinetic data is summarized in From these results, we can conclude that after the addition of porphyrin, the electron transfer is enhanced and the rugosity factor is increased, compared to CRGO.

Fig. 1.16. Cyclic voltamograms for glassy carbon electrode covered by (A) H2TPP-1CP/CRGO, (B) H2TPP-4CP/CRGO obtained in 0.1 M KCl with 1 mM benzoquinone in the range of potential from -0.4 to 0.2 V at various scan rates in the range 0.05-1 V s -1 , and The (C) show the variation of anodic and cathodic current peaks vs. v 1/2

The thickness of H2TPP-1CP/CRGO is much higher than that of H2TPP-4CP/CRGO, even which is much larger, indicating that H2TPP-1CP and CRGO were associated through π-π interaction between phenyl group and π-conjugated CRGO, which presumably induce the carboxylic group with carbon chain upwards. However, for H2TPP-4CP/CRGO, due to the formation of hydrogen bond, the thickness is less than the value of H2TPP-1CP/CRGO, which has no effect on the electron transfer of redox marker p-benzoquinone/hydroquinone. We can propose a mechanism of the H2TPP/CRGO association where the H2TPP-1CP and H2PTT-4CP were associated through π-π interaction of phenyl group with the graphene sheet specialy in the planelike edge. This could be presented in scheme 1.6.

103 Scheme 1.6. Schematic representation of interaction between H2TPP and CRGO

Interaction between metallporphyrins and CRGO

In order to further investgate the coverage of porphyrin, the metallporphyrin where manganese was chosen as metallic cation due to its reversible was monitored through electrochemical signal response of the Mn 3+ /Mn 2+ redox complex.

Molecular characteristics

Association of MnTPP-1CP/CRGO composite was evaluated by various methods, including UV-visible and FT-IR, as previously tested for H2TPPs/CRGO.

The UV-visible absorption of the MnTPP-1CP/CRGO composite in DMF exhibits a Soret band at 470 nm characteristic of the  electronic transition of metal complex and the two Q bands (569 and 607 nm) (Fig. S1.1). This result underlines that the association of the metalloporphyrin with graphene remains unmodified conformation of the porphyrin once adsorbed onto graphene surface. Fourier transform infrared spectroscopy (FT-IR) was also used to characterize the MnTPP-1CP/CRGO nanomaterial (Fig. S1.2). The FT-IR spectra of MnTPP-1CP/CRGO exhibit a large band at 3250 cm -1 which is assigned to OH vibrations of carboxylic group of modified 104 porphyrin and two stretching vibration bands in the double band region, at 1679 cm -1 corresponding to the C=O vibration and at 1592 cm -1 that can be attributed to C=C vibration. In comparison with the FT-IR spectrum of MnTPP-1CP, these two latter bands shift to lower wavenumber values. These results indicate that MnTPP-1CP interacts tightly with the conjugated aromatic structure of CRGO through π-π stacking and consequently this decreases the distance between CRGO and MnTPP-1CP.

Electrochemical characteristics

Beside the aromatic properties of MnTPP-1CP which enable strong interaction with CRGO, the presence of manganese complex exhibits interesting redox properties that could be used for the characterization of intrinsically properties of hybrid nanomaterial MnTPP-1CP/CRGO in phosphate buffer. Thus, a modified electrode was prepared by drop-coating a solution of MnTPP-1CP in DMF (0.1 mg/mL) onto CRGO-glassy carbon electrode surface. After drying, the modified surface was analyzed by square wave voltammetry (SWV), cyclic voltammetry (CV), and electrochemical Impedance (EIS) in PBS solution (Fig. 1.17 (

) 4
Where Q is the charge under the cathodic or anodic wave, n is number of electrons involved in the redox process, F is the Faraday constant, and A is the area of the electrode. On the basis of equation 4, we calculated that the average surface coverage of MnTPP-1CP is 3.34 ± 0.13 nmol cm -2 . These results agree with those obtained for a monolayer formation on an electrode surface 20 . Fig. 1.17B.b displays 105 the electrochemical signal of the manganese porphyrin after simple adsorption on the electrode surface. In this case, the average surface coverage was estimated to be 0.78 ± 0.07 nmol cm -2 , which was almost 4-times lower than the result obtained from hybrid nano-material MnTPP-1CP/CRGO electrode. All these results demonstrate i) the strong  stacking interactions between CRGO and the manganese porphyrin and ii) the ability of CRGO to immobilize a large amount of MnTPP-1CP. Furthermore, the combined porphyrin-graphene framework exhibits an excellent conductivity and a good electron transfer that both contribute to amplify the redox signal of metalloporphyrin.

Thanks to the properties of metalloporphyrin, the conditions of biosensor construction will be optimized and detected in detail next chapter.

Conclusion

In conclusion, tetraphenylporphyrins functionalized with one and four carboxylic groups were successfully synthesized by a simple and effective way in our work.

Meanwhile, the synthesized different types of graphene has been performed. The CV data following Nicolson model shows a value of as 0.01 cm s -1 which is in the same order of magnitude of carbon pyrolysed at 800 ℃ (0.018 cm s -1 ) electrode. The associations of H2TPPs bearing one and 4 carboxylic groups were also investigated.

We demonstrate that the association between H2TPP-1CP and CRGO is performed through π-π interaction between phenyl groups and graphene nanosheet with higher thickness, compared to the H2TPP-4CP with more negative charge of carboxylic group which is favored to anchoring at the edge of the graphene and forms hydrogen bond with hydroxyl group. The dispersion of CRGO is efficiently enhanced with the association process. Meanwhile, the addition of porphyrin has no effect on the ks compared to CRGO, which is up to 0.02 cm s -1 and 0.01 cm s -1 , respectively for H2TPP-1CP/CRGO and H2TPP-4CP/CRGO. Thus we demonstrate that the noncovalent interaction between H2TPP and CRGO leads to not only introduce the functionalization of CRGO and introduction of carboxylic group onto CRGO surface, but also retain their structural and electrochemical properties of CRGO. The incorporation of manganese (III) is favor to further investigate properties of the nanomaterials through its electrochemical signal variation of the Mn 3+ /Mn 2+ redox complex. The ability of CRGO facilitated to immobilize a large amount of porphyrin. This compound was synthesized in one step from the diphenylporphyrin according to the literature as described in a previous paper. 21 5 Porphyrine (4) (30 mg, 0.04 mmol) was dissolved in 10 mL of DMF in presence of a large excess of the corresponding ionic metal salts, Mn(OAc)2. The reaction was then running two days under argon at 80°C. The DMF was removed and CH2Cl2 was added. Then organic phase was washed many times with water and after evaporation of the solvent. The distilled pyrrol (5.6 mL, 1 eq.) dissolved in 20 mL propionic acid was added into p-methoxybenzaldehyde (9.75 mL, 1 eq) dissolved in 300 mL propionic acid under argon. The crude solution was reflux during 1.5 h at 140 ℃. DDQ was added to oxidize the crude sample. The solution was then evaporated and purificated. The functionalization of phenyl ring with a carboxylic group allows the attachment of single-stranded amino DNA (ssDNA) on the complex through a covalent bond. The manganese (Mn) has been chosen as metallic cation, due to its reversible and electrochemical signal response that will allow the electrochemical characterization.

The strategy followed for the DNA sensor construction was a step by step method

Instruments and measurements

Scanning Electron Microscopy (SEM) images were acquired using a ZEISS SUPRA TM 55VP GEMINI®.

Atomic Force Microscopy (AFM).

A commercial dimension 3100 (Vecco Instruments, USA) atomic force microscope was used for topographical characterization of the samples.

All measurements were performed at the tapping mode using rectangular silicon AFM tip.

UV-visible spectra were recorded with uvikon xl spectrometer.

Fourier Transform infrared spectra (FT-IR) were measured using a Bruker IFS66 FT-IR spectrometer equipped with a MCT detector and an attenuated total

Construction of DNA sensor

The CRGO solution was prepared by dispersing 1 mg of CRGO in 1 mL DMF, and ultrasonicated for 1h. The 1 µL of the prepared CRGO solution was drop-coated onto the electrode surface followed by drying step under the lamp. A drop of 1µL of a various concentration 0.01, 0.05, 0.1, 0.5, 1 mg/mL MnTPP solution in DMF was deposited onto the CRGO/GCE and the modified surface was then dried and rinsed with distilled water to remove non-specific adsorptions. Finally, the MnTPP-1CP/CRGO was activated in a freshly prepared 10 mM PBS solution (pH=7.4) of 20 mM EDC and 32 mM NHS. The activation step was running 1h at room temperature followed by immersing the electrode into a solution of 1µM ssDNA for 1h at room 124 temperature. Subsequently, the obtained DNA sensor was thoroughly rinsed with distilled water and stored in PBS at 4°C before use.

Target detection

The

Fig. 2.1. (A)

The SWV measurements in PBS, scan rate 12.5 mV.s -1 for 0.1 mg ml -1 MnTPP-1CP and 1 mg ml -1 CRGO modified electrode (line), after ssDNA 126 immobilization (dashed line), and after hybridization of 1μM target (dotted line); (B) Histogram of the relative variation of current peak of SWVs for DNA sensors after detection of 1 μM target.

b) The addition of NHS in coupling

The activated conditions in coupling reaction have also been optimized. The

Fig. 2.2. (A) The effect of the addition of NHS, and (B) the concentration of EDC c) Hybridization time

The hybridization time was optimized by EIS measurement in the presence of fixed concentration DNA target (1 fM) at various time from 20 min to 60 min (Fig.

Construction of the DNA sensor

Single-stranded DNA (ssDNA) was covalently interacted with metalloporphyrin through an amide link. The ssDNA labelled with an amine group with 6 carbonyl spacer (C6 amino link) are able to react with carboxylic groups present in metalloporphyrin immobilized on the CRGO surface. The reaction was performed in presence of EDC and NHS, as activating agents for one hour. After washing the modified electrode with distilled water to remove any reactants left, the surface was analyzed in buffer solution.

The coupling reaction was confirmed by FT-IR measurements and the FT-IR spectra of the ssDNA/MnTPP-1CP/CRGO surface (Fig. S2.1) exhibits the appearance of some new peaks at 3000 cm -1 , 1757 cm -1 , 1584 cm -1 and at 956 cm -1 which correspond to C-H, C=O, C-N and phosphates stretching, respectively confirming the presence of acids nucleic on the surface. These results suggest the covalent interaction 128 between ssDNA with MnTPP-1CP/CRGO on the electrode surface.

The formation of the electrochemical DNA sensor was then monitored by square wave voltammetry in PBS buffer thanks to the electrochemical signal response of Mn (Fig. 2.4A). SWV shows a decrease of the current, at -0.56 V corresponding to the reduction of Mn(III) to Mn (II) complex, after the immobilization of ssDNA. As it has been previously observed 1 , this phenomenon is explained by the slow diffusion of ions to the surface during the redox process due to the attachment of large ssDNA molecules on the electrode surface.

Selectivity and reproducibility

To The Rct/ Rct,ssDNA values were calculated to be 64%, 27% and 9% after hybridization with cDNA, cDNA with one-base mismatched and non-complementary sequences, respectively. This indicates that the DNA sensor has a good hybridization selectivity to discriminate the complementary from the non-complementary sequences.

Nethertheless, this selectivity might be improved by adding more negative charges on 133 the electrode surface. Indeed, the low variation observed for the non-complementary DNA could be attributed to the non-specific adsorptions of the DNA onto electrode surface. Increasing the number of negative charges on the surface might limit the interaction of the negatively charged ssDNA sequences by electrostatic repulsion. For example, the introduction of more carboxylic functions on phenyl groups of the metalloporphyrin might improve the selectivity to the target sequences detection.

The reproducibility of the DNA hybridization platform was tested by four impedance measurements with four independent biosensors. Each sensing layer was freshly prepared. A relative standard deviation (RSD) of less than 3% for the Rct/ Rct,ssDNA value was estimated, which showed the high reproducibility of the biosensor layer.

Conclusion

In conclusion, based on a π-conjugated framework composed of a manganese (III)

tetraphenylporphyrin and graphene, we design the first example of electrochemical DNA sensor for highly sensitive and selective detection of DNA target. We have demonstrated the different advantages of using the manganese tetraphenylporphyrin in such material. The manganese tetraphenylporphyrin was functionalized with one carboxylic group for covalently interaction with ssDNA on the GC electrode surface which offers a stable sensing layer. The incorporation of a redox metal inside the macrocycle ring of porphyrin allows the electrochemical characterization of the film.

EIS measurement demonstrates that the Rct value of the MnTPP-1CP/CRGO hybrid nanomaterial increases after ssDNA immobilization and hybridization to its complementary sequences, which is used as a sensing signal. A detection limit of 2×10 -16 M is estimated with a moderate selectivity that could be improved by introducing more negative charge function on the porphyrins, thanks to their chemical modification ability. These results demonstrate that MnTPP-1CP/CRGO platform provided a very good candidate for direct DNA sensor and are very promising in DNA sensors applications.

Instruments and measurements

Atomic Force Microscopy. A commercial dimension 3100 (Vecco Instruments, USA) atomic force microscope was used for topographical characterization of the samples. All measurements were performed at the tapping mode using rectangular silicon AFM tip.

room temperature. Subsequently, the obtained DNA sensor based on H2TPP-1CP/CRGO was thoroughly rinsed with distilled water and stored in PBS at 4 °C before use. After each step of construction of the DNA sensor, the modifications of the electrode surface were controlled by SWV method. The DNA sensor based on H2TPP-4CP/CRGO was constructed according to the same procedure as that based on H2TPP-1CP/CRGO.

DNA Target detection

The DNA hybridization of complementary 25-bases targets with ssDNA probe associated to the modified GCE surface was performed by immersing the modified electrode in solution of various concentration of DNA target of hepatite C gene for 40 min at 47 º C. The target concentrations are increased successively from 1 aM, 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM to 1 nM. After each incubation, the modified electrode was washed with distilled water for three times to eliminate no hybridized single strand DNA and then analyzed by SWV method. The detection of non-specific interactions was conducted according to the same procedure described previously, by immersing the modified electrode in a PBS buffer solution containing different concentration of non-complementary DNA target in from 1 aM, 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM to 1 nM. To check the reproducibility, the experiment was repeated four times with freshly prepared DNA sensors individually.

PCR sample detection

The DNA from PCR samples was denaturized by heating at 95 ℃ for 5 min, and then immersed in an ice bath for 1 min S3.1). After ssDNA interaction, an obvious increase of the electron transfer resistance (Rct2, Table S3.1) from 59 to 166 kΩ is observed and confirms that the successful covalently attachment of ssDNA onto H2TPP-1CP/CRGO layer. This variation can be explained by a blocking of diffusion ions process to the electrode surface. The thickness of the layer is sharply deceased from 72 to 31 nm (Table S3.1) and the N2 value (Table S3.1) which reflects the roughness of the surface remains In order to further evaluate the electronic properties and construction of the ssDNA/H2TPP-1CP/CRGO surface, CV and EIS measurements were performed in 5 mM [Fe(CN)6] 3-/4-species (Fig. 3.2). Fig. 3.2A shows that after the immobilization of ssDNA, the redox peaks of [Fe(CN)6] 3-/4-couple have separately shift to the irreversible trend, meanwhile the Rct estimated remains constant (from 3.49 to 3.56 kΩ, Table S3.2). This result can also suggest that the immobilization of ssDNA tends to lie on the hybrid surface through layer by layer, which has a little influence on the electric properties of the surface. by the slope of the curve Ep versus v -1/2 (Fig. S3.1). These parameters have been calculated for both [Fe(CN)6] 3-/4-and benzoquinone/hydroquinone redox couples before and after ssDNA immobilization. All the results are presented in Table 3.1.

For both redox couples, D0 decreases sensibly after ssDNA immobilization, from 6×10 -6 to 4.6×10 -6 cm 2 s -1 in [Fe(CN)6] 3-/4-solution and from 1×10 -6 to 0.5×10 -6 cm 2 s -1 in the neutral redox marker p-benzoquinone. This confirms that the successful interaction between ssDNA and hybrid nanomaterial which is in agreement with the result of EIS in PBS. The diffusion process of redox molecules in solution to the electrode surface becomes more difficult due to the presence of the ssDNA layer on the modified electrode. by SWV experiments at Ered = -0.1 V, a variation (ΔQ) is calculated to be 0.029 mC.

Table 3.1. Summary of the diffusion coefficient (D0) and ks of the redox marker [Fe(CN)6]

A variation of the surface coverage (Γ) can be calculated according to the equation:

Γ = Q / nFA (Eq.2)
, where n is the number of electrons involved in the redox process, F the Faraday constant, and A the area of the electrode. The decrease of the surface coverage of benzoquinone (2.2 ± 0.65 nmol cm -2 ) reflects the immobilization of ssDNA on the H2TPP-1CP/CRGO layer.

-0.4 -0.3 -0.2 -0.1 0.0 0.1 species to the electrode surface. When ssDNA hybridized with cDNA target, doublehelix structure is formed and electronegative phosphate groups are introduced on the surface. It is more difficult for [Fe(CN)6] 3/4 to reach the electrode due to the electrostatic repulsion. Hence, the Rct value increases with the formation of doublestranded DNA (Fig 3 .4A). Hybridization reaction was also performed by CV measurements in [Fe(CN)6] 3/4-solution (Fig. 3.4B). The current of [Fe(CN)6] 3/4-is decreased and the reversibility of redox process is lost with the increasing of the target concentration. These results are in good agreement with those obtained in EIS experiments. The evaluated logarithmic relationship between ΔRct/Rct ssDNA and the concentration of the cDNA target was shown in the inset of Fig. 3.4A. The ΔRct corresponds to the difference between Rct value of the ssDNA linked to the electrode surface and that after hybridization with cDNA (ΔRct =Rct,dsDNA -Rct,ssDNA). The regression equation was ΔRct/Rct,ssDNA = 0.1642 lg Ctarget +2.77 with regression coefficient of 0.98. The detection limit was estimated to be 7 × 10 -14 M (signal-tonoise ratio of 3 independent measurements, taking into accord the standard deviation of 0.2136).

Fig. 3.5. SWV experiments during detection of biosensors based on H2TPP-1CP/CRGO at various concentration of targets (1 aM to 1 nM).

The relative changes of current peak versus the concentration of complementary ssDNA targets were shown in Fig. 3.6. The detection limit was estimated to be 2 × 10 - 18 M for DNA sensor based on H2TPP-1CP/CRGO, (signal-to-noise ratio of 3 independent measurements, taking into accord the standard deviation of 0.0101), which is lower by four orders of magnitude compared to that estimated in [Fe(CN)6] 3- /4-solution for the same DNA sensor.

Selectivity of DNA sensor

To test the selectivity of the DNA sensor based on H2TPP-1CP/CRGO, non- S3. 4). An obvious increase from 72 to 154 kΩ of the electron transfer resistance after immobilization confirms as well the successfully covalently attachment of ssDNA to H2TPP-4CP/CRGO composite. This variation can still be explained by a blocking of diffusion ions process to the electrode surface. The thickness is deceased from 20 to 6 nm (Table S3.4) and the N2 (that reflects the roughness of the layer) varies from 0.56 to 0.64. These results indicate that the covalently attachment of ssDNA on the hybrid nanomaterial surface affects the molecular organization on the surface. This can explain the decrease of the thickness of the nanomaterial layer as it was also observed with the previous DNA sensor The interaction between H2TPP-4CP/CRGO and ssDNA has an apparent effect on the electronic properties of the modified surface, presumably due to a rearrangement of the orientation of the biomaterial to the electrode surface induced by the charge repulsions that lead to modifications of the electronic charges on the surface. Fig. 3.9 shows the SWVs performed after the construction of DNA sensor exhibiting a decreasing of current after ssDNA covalently interacted with H2TPP-4CP/CRGO surface. The variation of ΔQ is calculated to be 0.06 mC and the calculated surface coverage of reduced benzoquinone is evaluated as 4.6± 0.43 nmol cm -2 which is around 2 times higher compared to that of the H2TPP-1CP/CRGO modified electrode. This demonstrates that with the same initial concentration of porphyrin, a largest amount of ssDNA is immobilized on the surface of the H2TPP-4CP/CRGO. This result is not surprising since the number of reactive carboxylic functions has also increased on the surface. The diffusion coeffcient (D0) of the redox marker [Fe(CN)6] 3-/4-in aqueous media was calculated by the Randles-Sevcik equation (Table 3.2) following the same procedure as H2TPP-1CP/CRGO used. It was estimated to be 2.5×10 -6 cm 2 s -1 before and 4×10 -6 cm 2 s -1 after the attachment of ssDNA on the nanomaterial. The D0 increases sensibly which is in good agreement with CV and EIS observations (better reversibility of the redox system and better conductivity of the nanolayer). In pbenzoquinone solution, the neutral redox marker, D0 was also calculated and was found to be 5.6×10 -6 cm 2 s -1 before and after the immobilization of ssDNA, the value decreases to 3.8×10 -6 cm 2 s -1 (Table 3.2). In this case, the diminution of D0 can only be explained by steric effects due to the presence of the ssDNA anchored on the surface, that blocks the diffusion of ions to the electrode, instead of charge repulsions because the redox marker is neutral. A larger amount of ssDNA immobilized on the modified surface, presumably due to the covalently interaction between hybrid complex and ssDNA tends to To eliminate the effect of electronic repulsions, the electrochemical detection of DNA targets was monitored by SWV measurements in 1 mM benzoquinone. This result is in the same order of magnitude than the variation observed during the detection of 1 fM of nc DNA taget presented in the histogram (Fig. 3.12 B). Comparing to the electronic properties recorded in two different redox markers, a negatively charged [Fe(CN)6 3-/4-] and a neutral one (benzoquinone), we noticed some real difference of the two bio-nanomaterials, in term of thickness, configuration of ssDNA on H2TPP-nCP/CRGO layer and the repartition of charges on the surface. The measurement data were summaried in Table 3.3.

The decreased of coverage of benzoquinone which is reduced on the ssDNA/H2TPP-4CP/CRGO modified electrode is 2 times higher compared to that of ssDNA/H2TPP-1CP/CRGO, demonstrating that a larger amount of ssDNA is immobilizated on the surface of the H2TPP-4CP/CRGO.

In solution of [Fe(CN)6 This observation is supported by the increase of the relative diffusion value [ΔD0/D0 = 60%] of Fe(CN)6 3-/4-ions and the reversibility of the redox marker signal (Fig. 3.8).

Although a larger amount of ssDNA is immobilized on the surface, the D0 increases, suggesting that the ssDNA is covalently interacted with hybrid complex on the edge of the CRGO instead of on the stacking basal plane. Indeed, we proposed in the chapter Ⅰ, that the H2TPP-4CP interacted with CRGO on its edge. Furthermore, the thinner thickness of 6 nm could also support the hypothesis. In contrast, the decreased of ΔD0/D0 after ssDNA immobilization of H2TPP-1CP/CRGO nanomaterial indicates that the ssDNA lie on the surface of hybrid nanomaterial, layer by layer, which block the diffusion of the Fe(CN)6 3-/4-ions to the surface. A higher thickness of 30 nm also confirms this result.

In p-benzoquinone solution, eliminating the effect of the charge, difference are also noticed and are in good agreement with the organisation of ssDNA we proposed.

Compared to H2TPP-1CP/CRGO, a lower decrease of relative value of ΔD0/D0 and Δks/ks are observed for H2TPP-4CP/CRGO nanomaterial after reaction, in spite of a larger amount of ssDNA immobilized on the modified surface. As we think, it is presumably due to the covalently interaction of ssDNA with hybrid nanomaterial on the edge of the CRGO, and thus leads to release the surface and facilitates the diffusion of the ions to the electrode surface. To further characterize hybrid nanomaterials and their morphology, AFM images were performed for ssDNA/H2TPP-1CP/CRGO layer (a) and ssDNA/H2TPP-4CP/CRGO (b) after ssDNA immobilization (Fig. 3.13). As it is shown, the morphology is completely different compared to the two DNA sensors: a surface more dense and compact for picture a) and a surface with helical elements for picture b). In the latter case, the helical structures could be attributed to the single-strand structure of ssDNA, grafted certainly on the edge of the hybrid complex. In contrast for ssDNA/H2TPP-1CP/CRGO, the surface is more compact due to probably an immobilization of ssDNA layer by layer, homogeneously, on the plane hybrid surface layer. In benzoquinone solution by SWVs analysis, DNA sensor based on H2TPP-4C/CRGO exhibits a higher LOD than the other but the linear range is sensibly in the same order of magnitude for the two sensors even if the amount of ssDNA immobilized on the surface of the H2TPP-4C/CRGO is larger. Thus, the selectivity, tested in neutral redox marker for the detection of 1 fM of non-complementary target (Fig. 3.14, deduced from the results of Fig. 3.6 and 3.12), exhibits a better response for the DNA sensor based on H2TPP-4CP/CRGO, with only 15% of variation for the relative change of peak current compared to 30% for the DNA sensor based on H2TPP-1CP/CRGO. This difference can be explained by the presence of more negative charges on the surface, due to larger amount of carboxylic functions on porphyrins, preventing so the non-specific interactions with DNA sequences to the surface. However, this result was improved but not completely satisfactory.

The DNA sensor based on the H2TPP-4CP/CRGO hybrid nano-material demonstrated better performance and was used to further explore in detecting PCR samples.

Instruments and measurements

Atomic Force Microscopy (AFM). A commercial dimension 3100 (Vecco Instruments, USA) atomic force microscope was used for topographical characterization of the samples.

All measurements were performed at the tapping mode using rectangular silicon AFM tip.

Fourier Transform infrared spectra (FT-IR) were measured using a Bruker IFS66 FT-IR spectrometer equipped with a MCT detector and an attenuated total reflectance (ATR) crystal of germanium.

Electrochemical measurements were performed using an AUTOLAB PGSTAT

181

A drop of 2 µL of the prepared 0.1 mg mL -1 H2TPP-4CP/CRGO suspension was deposited onto the -PEA modified glassy carbon electrode surface followed by drying step under the lamp, and rinsing with distilled water. Finally, the H2TPP-4CP/CRGO was activated in a freshly prepared 10 mM solution (pH=7.4) PBS of 20 mM EDC, 32 mM NHS. The activation step was running for 1h at room temperature.

Then the electrode was immersed in 1µM solution of ssDNA probe prepared in PBS buffer for 1h at room temperature. Subsequently, the obtained DNA sensor based on H2TPP-4CP/CRGO/PEA was thoroughly rinsed with distilled water and stored in PBS at 4 °C before use. After each step of construction of the DNA sensor, the surface modifications were controlled by SWV method in benzoquinone.

Target detection

The electrode is evaluated to be as 0.106 mC compared to 0.215 and 0.240 mC for 1 β-PEA/GCE and 2 β-PEA/GCE, respectively, confirming that less benzoquinone species are reduced with such layer (Table 4.2).

After the interaction of H2TPP-4CP/CRGO nanomaterial, the reduction potential of benzoquinone shifts positively to -0.12 V for the three modified electrodes H2TPP- As it can be seen in Fig. 4.4a, the 1 β-PEA /GCE surface exhibits a disordered topography surface where PEA is electrodeposited irregularly on the electrode surface with some free-space of glassy carbon. The nanomaterial H2TPP-4CP/CRGO (Fig. From calibrations curves, the sensor exhibiting the best performance appears to be the sensor based on H2TPP-4CP/CRGO/ 2 β-PEA material in term of LOD (1.2 10 - 16 M) and linear range [10 -18 -10 -9 M] (Table 4.3, Fig. S4.2). We also noticed that the linear range is higher with a material based on -PEA layer and it increases with the increasing of its concentration to then, decreases.

In view of all these results, the most ordered and uniformed self-assembled surface (H2TPP-4CP/CRGO/ 2 β-PEA) leads to the sensor with the best performance. ssDNA/H2TPP-4CP/CRGO/4 β-PEA 1.2×10 -18 10 -18 ~10 -14 Rct,ssDNA values were calculated to be 27% and 9% respectively. This undesired variation observed after the non-complementary DNA interaction could be attributed to some non-specific adsorptions of the DNA onto the electrode surface. Increasing the number of negative charges on the surface should limit this kind of interactions by charge repulsions and so should improve the DNA sensor performance.

Selectivity and reproducibility

Thus, a designed electrochemical DNA sensor based on π-conjugated nanomaterial composed of chemically reduced graphene oxide (CRGO) and tetraphenylporphyrin functionalized with numerous carboxylic groups (H2TPP-nCPn=1, Keywords : nanomaterials; porphyrins; graphene; sensitive; selective; biosensor Abstract : The design and development of biosensors for direct detection of biomarkers in biological fluid sample is real challenge in research for their application in point of care system in diagnostic. My project is focus on the elaboration of a new platform for biological detaction composed of graphene which exhibits a high electroactive and specific surface suitable for grafting numerous biomoleculer with metalloporphyrins as a novel redox marker for following biological recognition process obtained in the case of DNA sensor. The concept will be to demonstrate that the metaloporphyrines where there redox potential could be controlled by the nature of metal ions could be applied in multidetection electrochemical biosensor platform.

The association of metalloporphyrin with graphene will offer numerous advantages, as promoting electron transfer reaction and having a large surface for protein immobilization. The detection of target present will be performed following redox activity of the metalloporphyrins grafted on the surface.

We have already synthesized various modified metalloporphyrins and characterizatized by usual spectrometry techniques such as NMR, UV and MS. The DNA biosensor has been constructed by the composite of graphene and metalloporphyrin and DNA probe as bioreceptor following covalent attachment approach and optimization of the biosensor construction for improving stability and reproducibility.

And we have already done some detection of DNA Target by various electrochemical methods including cyclic voltammetry, square wave voltammetry and impedance spectroscopy. This strategy was performed firstly with on Mn inserted porphyrin and then generalized to various metal complex for multidetection approach. Meanwhile, the hybrid nanomaterials which combined the features of both graphene (high conductivity and large specific surface area) and porphyrins (physical and chemical properties and electron transfer ability), such as tetraphenylporphyrin bearing one and four carboxylic group named H2TPP-nCP(n=1, 4) and tetraphenylporphyrin incorporating manganese (III) (MnTPP-1CP) were successfully synthesized. The interaction modes between H2TPP-nCP(n=1, 4) and chemically reduced graphene oxide (CRGO) was studied in my thesis work. To investigate the characteristic properties of as-prepared nanomaterials, scanning electron microscopy (SEM), atomic force microscopy (AFM), UV-visible spectra, Fourier Transform infrared spectra (FT-IR), X-ray photoelectron spectra (XPS). In order to eliminate the non-specific absorption, the modification of the electrode by monolayer PEA was also applied and obtain excellent application for the detection of DNA target

  .......................................................................................................................................... RESUMÉ ........................................................................................................................................................... GENERAL INTRODUCTION ............................................................................................................................ PART A: BIBLIOGRAPHY Results and Discussion .............................................................................................................................. 1. Electrodeposition of β-PEA .................................................................................................................. 2. Characterisation of self-assembled H2TPP-4CP/CRGO/β-PEA ............................................................. 3. Construction of the DNA sensor .......................................................................................................... 4. Detection of DNA target ...................................................................................................................... 5. Selectivity and reproducibility ............................................................................................................. Conclusion .................................................................................................................................................. Supporting Information ............................................................................................................................. GENERAL CONCLUSIONS ........................................................................................................................... ABBREVIATIONS ........................................................................................................................................ RESUMÉ Les biocapteurs é lectrochimiques sont une classe de capteurs trè s5 prometteurs pour l'identification et la quantification de biomolé cules en solution. En effet, cet outil d'analyse présente un certain nombre de propriétés intrinsèques qui en font d'eux des outils très attractifs et prometteurs dans des domaines aussi variés que le médical, l'agroalimentaire, l'environnement…. Ils possèdent une grande sensibilité dans leur réponse (jusqu'à l'attomole détecté) en temps réel et directe, une facilité de mise en oeuvre, à faible coût et la possibilité de les miniaturiser et les inté grer dans des é quipements transportables. Les biocapteurs sont classé s en trois caté gories selon la nature des biomolé cules immobilisé es à la surface ou des ré actions é tudié es. On peut citer les immunocapteurs, les capteurs enzymatiques et les capteurs d'ADN. Dans le cadre de ma thè se, je me suis inté ressé e à dé velopper des capteurs d'ADN capables de détecter deux maladies infectieuses, l'hé patite C et la tuberculose, en ciblant des échantillons d'ADN synthétiques et PCR L'infection par le virus de l'hé patite C (VHC) est un problè me de santé mondial qui a touché environ 170 millions de personnes dans le monde et constitue l'une des principales causes de dé cè s lié s à une cirrhose hé patique et à un carcinome hé patocellulaire. L'importance des tests de diagnostic molé culaire à faible coût est particuliè rement importante pour les pays en dé veloppement car ils sont dé jà accablé s par un nombre croissant de patients atteints d'hé patite C qui sont gé né ralement é conomiquement en arriè re. L'avè nement des approches diagnostics molé culaires a permis de dé velopper des analyses d'acide nuclé ique plus sensibles et spé cifiques que les technologies à base d'anticorps. La tuberculose (TB) est causé e par le bacille à l'acide Mycobacterium tuberculosis (Mtb) et pré sente l'un des taux d'infection les plus é levé s au monde touchant 1 personne sur 3 et 1,5 million de dé cè s en 2013. Dans les pays les plus pauvres, le plus grand obstacle qui subsiste est la difficulté à dé tecter le bacille en dehors des lieux hospitaliers. Par consé quent, il est né cessaire de disposer d'un nouvel outil de diagnostic qui puisse ê tre dé ployé rapidement et facilement dans des zones é loigné es à forte charge de TB, et à faible coût en comparaison des mé thodes de diagnostic conventionnelles.

  4CP/CRGO/2β-PEA et ADN/H2TPP-4CP/CRGO/4β-PEA. Cette ré ponse du signal est caractéristique d'un signal on. De plus, le capteur ADN/H2TPP-4CP/CRGO/2β-16 M-10 -9 M] et une limite de dé tection de 10 -19 M. Une é tude de la sensibilité des capteurs en présence du brin d'ADN non-complé mentaire a montré une trè s faible, voire absence de ré ponse é lectrochimique des capteurs ADN/H2TPP-4CP/CRGO/2β-PEA et ADN/H2TPP-4CP/CRGO/4β-PEA, mê me pour des concentrations élevées de l'ADN non complémentaires. Ainsi l'incorporation d'une couche auto assemblée sur la surface, avec une concentration supérieur à 2mM, a permis l'absence de toutes adsorptions non spécifiques de l'ADN sur la surface du capteur. Il reste né anmoins à ré aliser des é tudes de dé tection du capteur ADN/H2TPP-4CP/CRGO/2β-PEA en milieu biologique pour é valuer sa performance.

  response. Electrochemical DNA sensors become an attractive technique for the identification of biomolecules due to miniaturization possibilities, low cost and direct lecture of electric signals. However the choice of a transducer, which affords an electrochemical signal, is crucial in biosensor construction. Performance of DNA sensor is characterized by some important parameters such as dynamic and linear range of detection, limit of detection (LOD), sensitivity, and selectivity. During my thesis, developing efficient conducting transducers combining the porphyrins functionalized with various groups and graphene for high sensitive and selective electrochemical detection was a main objective of my research. I worked on DNA detection and in the development of DNA sensors for two infectious diseases like hepatitis C with synthetic DNA targets and tuberculosis DNA from real PCR samples. Such examples were used to demonstrate that DNA sensor could be generalised to all infectious diseases and used in point of care system. These DNA sensors are especially attractive in the biological field and public health. I divided my thesis into two Parts, Part A, of which describe generally about electrochemical DNA sensor and transduces developed and explored in DNA sensors and Part B is related to the results and discussion of my work, which will be composed of four chapters.

  characterized through different analytical techniques such as FT-IR, UV and electrochemical measurements. The immobilization of ssDNA and hybridization with complementary sequences (cDNA) lead to the change of electrochemical properties for MnTPP-1CP/CRGO.The conditions for the constructive process of DNA sensor, including the concentration ratio of MnTPP-1CP and CRGO, the concentration of activated reactants (EDC and NHS), and the hybridization time was also to be optimized by various electrochemical tests. Selectivity of the DNA sensor was investigated in presence of non-complementary and one-mismatched DNA sequence. A detection limit was estimated and could be improved by introducing more negative charge function on the porphyrins, thanks to their chemical modification. The selectivity was expected to be improved by adding more negative charges on the electrode surface due to the charge repulsions.Thus, the purpose in chapter Ⅲ was to focus on DNA sensor based on the H2TPP-nCP/CRGO hybrid nanomaterials (with n=1 or 4) covalently attached with single-strand DNA to afford ssDNA/H2TPP-nCP/CRGO. The layer was characterized by Atomic Force Microscopy (AFM) and electrochemical methods. I wanted to present novel electrochemical DNA sensor based on self-assembled nanomaterial of a π-electron-rich conjugated structure of CRGO associated with two types of -conjugated H2TPP-1CP and H2TPP-4CP, respectively. Porphyrin molecules are versatile compounds that are easily functionalized with numerous designed carboxylic groups that could covalently interact with a 5'-amino singlestrand DNA (ssDNA) through stable amide link. The electronic properties of such nanomaterials was studied through square wave voltammetry (SWV), electrochemical impendence spectra (EIS), and cyclic voltammetry (CV). On respectively employing a negatively charged redox marker, [Fe(CN)6] 3-/4-and a neutral one, p-benzoquinone, the pattern of interaction between H2TPP-nCP/CRGO and ssDNA could be envisaged by means of the calculated relative change of diffusion coefficient (D0) and homogenous electron constant (ks). The DNA immobilization and hybridization with complementary sequences

Fig. 1 .

 1 Fig. 1. The first commercial biosensor developed by Professor Leland C Clark

Fig. 4 .

 4 Fig. 4. Schematic representation of detection by enzyme labelled DNA target

Fig. 5 .

 5 Fig. 5. Schematic illustration of the hybridization detection protocol.

Fig. 7 .Fig. 8 .

 78 Fig.7. Schematic representation of DNA sensors based on DNA labelled with Fc

Fig. 9 .

 9 Fig. 9.. Schematic presentation of biosensor based on redox properties of DNA and reaction of oxydation of guanine

  developed a highly sensitive and selective electrochemical DNA hybridization sensor (Fig.10) for breast cancer marker BRCA1 by using the sensitively label-free sensing technique of electrochemical impedance spectroscopy (EIS). Capacitance and electrical resistance of the electrode surface are the sensitive indicators and their modulations are related to the surface property variations. It is commonly known that the reasons for EIS response change are mainly owing to the charge effect and the steric hindrance impact. In order to verify the main reason for the EIS change after the hybridization, two redox markers were applied in these EIS tests, a positively charged complex [Ru(NH3)6] 3+ and a negatively 29 one [Fe(CN)6] 3-/4-. The increased Rct in negatively charged and decreased Rct in positively charged demonstrated that the charge effect due to the strong negative charge of the DNA target played a key role in the measured electrochemical EIS signal with redox markers.Significantly, this developed biosensor strategy achieved a linear detection range from 1.0 FM to 10.0 pM with a limit of detection of 0.3 fM, when negatively charged [Fe(CN)6] 3-/4-as indicator[START_REF] Bollo | Electrooxidation of DNA at glassy carbon electrodes modified with multiwall carbon nanotubes dispersed in chitosan[END_REF] are applied.

Fig. 10 .

 10 Fig. 10.. (A) Schematic illustration of Peptide SAM formation on Au electrode surface. (B) DNA sensor fabrication and sensing procedures

Fig. 12 .

 12 Fig. 12. Schematic diagrams of the detection of DNase I activity based on electrochemical method.

Fig. 15 .

 15 Fig. 15. Schematic illustration of the hybridization detection protocol

Fig. 16 .

 16 Fig. 16. Schematic illustration of PPy-PEDOT-Ag nanocomposite

Fig. 17 .

 17 Fig. 17. Different allotropes of carbon[START_REF] Atta | Graphene-A Platform for Sensor and Biosensor Applications[END_REF]

Fig. 18 .

 18 Fig. 18. Illustration for the construction and detection strategy of the FLN-based DNA sensor

Fig. 19 .

 19 Fig. 19. Schematic representation of the enhanced electrochemical detection of DNA hybridization based on the MWNTs-COOH constructed DNA biosensor

47

 47

Fig. 26 .

 26 Fig. 26. Schematic illustration of the graphene exfoliation process

3. 1 3 . 1 . 1

 1311 Different approaches to immobilize DNA onto reduced graphene oxide Through physical adsorptionDNA adsorption on graphene surface is well known and because of its high specific surface area, the carbon familly possesses potentially larger DNA adsorption capacities than other nanomaterials117 . Due to the nature of the nucleobases of DNA (aromatic skeleton, polar), nucleotides interact with reduced graphene oxide through several interaction forces like, hydrogen bonding, base staking, electrostatics, van der Waals and hydrophobic interactions. Besides, Song et al.118 has demonstrated that numerical simulations and experimental had confirmed that the ssDNA sequence could adsorb on graphene sheet surface with all nucleobases as unexpected. Indeed, the Fig.29illustrates the two modes of interaction of single-stranded DNA strand on two different surfaces: graphene -Au(111) surface (in green and yellow sphere respectively) and on a typical Au(111) surface (the figure inset).

Fig. 29 .

 29 Fig. 29. Schematic representation of an ssDNA nucleobase on the surface of the graphene-on-Au(111) material.

3. 1 . 2

 12 Through covalent attachment The presence of carboxylic acid and hydroxyl groups at the edges and on the basal plane of reduced graphene oxide are used to fix ssDNA sequence. Bonanni et al 119 could successfully functionalize graphene with ssDNA oligomers with carbodiimide chemistry (Fig 30). They showed that such DNA-graphene hybrid structures exhibit strong biorecognition ability.

Fig. 30 .

 30 Fig. 30. Covalent functionalization of graphene by ssDNA. a) Sufo-NHS esters formed on carboxylated graphene react with amino-modified ssDNA probes to form an amide bond; this results in, b) covalently func-tionalized graphene by ssDNA.

54

 54

Fig. 32 .

 32 Fig. 32. Schematic diagram of the electrochemical DNA biosensor.

Fig. 33 .

 33 Fig. 33. Proposed mechanism for the formation of stable aqueous dispersion of graphene.

Fig. 34 .

 34 Fig. 34. Schematic representation of graphene functionalization with PTCA, ssDNA immobilization, and hybridization

  formed is very large and the purification and separation workup are cumbersome. The latter one is the combination of pyrrole building blocks with aldehydes that can be used in a [2+2] or [3+1] condensation reaction 150 (path B). All these reactions involve acid-catalyzed condition reactions, often resulting in by product formations and limiting the types of substituents used. At least, the last strategy, developed by Senge et al. is to introduce different substituents directly at the meso-positions from trisubstituted porphyrines, for example 151 . The path A and B will be particularly focus on in the next parts.

Fig 37 . 2 . 1

 3721 Fig 37. General approach for the synthesis of porphyrin

Fig. 38 .

 38 Fig. 38. Formation of tetraphenylporphyrin (TPP) by Lindsey

64 Fig. 43 .

 6443 Fig. 43. Schematic representation of graphene-metalloporphyrin hybrid

Fig. 44 . 3 . 3

 4433 Fig. 44. Schematic diagram of photoelectrochemical sensor

Fig. 45 .

 45 Fig. 45. (A) Cyclic voltammograms (CVs) and (B) Electrochemical impedance spectroscopy (EIS) spectra of 10 mM Fe(CN)6 3-/4-in 1.0 M KCl at bare GCE, TCPP and TCPP-RGO modified GC electrode. Scan rate: 50 mV/s.

79 3. 1

 791 Synthesis of porphyrins 3.1.1 Synthesis of porphyrin functionalized with one carboxylic group

Scheme 1 . 1 .Scheme 1 . 2 .Scheme 1 . 3 .

 111213 Scheme 1.1. Synthesis of 5-(p-methoxyphenyl)triphenylporphyrin (i) 1) TFA and 2) DDQ, air

Fig. 1 . 1 .

 11 Fig. 1.1. The electrochemical exfoliation process

83 3 . 3 1 Fig. 1 . 3 .

 33113 Fig. 1.3. UV-Visible spectra of H2TPP-1CP, H2TPP-4CP and MnTPP-1CP measured in DMF

Fig. 1 . 4 .

 14 Fig. 1.4. X-ray photoelectron spectra of the GO before (A) and after (B) reductive reaction. , and (C), (D) the corresponding C1s XPS spectra.

Fig. 1 . 5 .Fig. 1 . 6 (

 1516 Fig. 1.5. Raman spectra of GO and CRGO

Fig. 1 . 7 .

 17 Fig.1.7. Images of DMF dispersions (1 mg mL -1 ) of CRGO, H2TPP-1CP/CRGO and H2TPP-4CP/CRGO. To characterize such hybrid nanomaterials and their properties, various physical and chemical methods were investigated. SEM and AFM images were performed for the characterization of morphologies. Spectroscopic characterization by UV-Visible, FT-IR and Raman were realized for molecular characterization and EIS, CV for electrochemical analysis to investigate electron transfer properties.

77 Fig. 1 . 10 .

 77110 Fig. 1.10. UV-Visible spectrum of (A) H2TPP-1CP and H2TPP-1CP/CRGO; (B) H2TPP-4CP and H2TPP-4CP/CRGO with successive increased comcentration of CRGO measured in DMF. The affinity constant calculation of H2TPP-1CP/CRGO (C) and H2TPP-4CP/CRGO (D)

39 Fig. 1 . 12 .

 39112 Fig. 1.12. Raman spectra of CRGO, H2TPP-1CP/CRGO and H2TPP-4CP/CRGO

99

 99

Fig. 1 . 13 .

 113 Fig. 1.13. Nyquists plots measured in PBS solution at applied potentiel -0.3V with Dc potential of 10mV at frequency range of 100 KHz to 0.01Hz with various modified electrode H2TPP-1CP/CRGO (A) and H2TPP-4CP/CRGO (B)

Fig. 1 . 14 .

 114 Fig. 1.14. Nyquists plots measured in [Fe(CN)6] 3-/4-solution at applied potentiel 0.15 V with various modified electrode (A)CRGO, H2TPP-1CP/CRGO and (B) H2TPP-4CP/CRGO.

  ). As shown by the square wave voltammogram (Fig. 1.17A), CRGO-based electrode covered with MnTPP-1CP exhibits in PBS, a pair of apparent peaks at -0.56 and -0.44 V vs Ag/AgCl (Fig. 1.17A.c) compared to CRGO electrode (Fig. 1.17A.a).These two peaks correspond respectively to the reduction of Mn 3+ to Mn 2+ at -0.56 V and the reverse reaction at -0.44 V. Surface coverage () of the manganese (III) tetraphenylporphyrin attached to CRGO can be measured from CV measurements (Fig.1.17B), following the equation 419 :

Fig 1 . 17 .Fig 1 . 18 .

 117118 Fig 1.17. (A) SWV measured with scan rate 12.5 mV.s -1 and (B) CV measurements in PBS at the scan rate of 20 mV/s for various modified GCE with CRGO (a), MnTPP-1CP (b), and MnTPP-1CP/CRGO (c)

  chemically reduced graphene oxide (CRGO) was characterized by XPS and Raman spectroscopy. The CRGO nanomaterial demonstrates high level of reduction. The electrochemical performance exhibits high electrochemical activity monitored by high reversibility of redox marker [Fe(CN)6] 3-/4-and high intensity compared to other carbon materials GO and GCE. The homogenous electron transfer ks calculated from

  methoxyphenyl)-10,15,20-triphenylporphyrin.(1)

3 .

 3 Synthesis of 5,10,15,20-[4-(carboxylbutyloxy)phenyl] tetraphenylporphyrin (H2TPP-4CP) 5,10,15,20--(4-methoxyphenyl) triphenylporphyrin. (5)

Fig. S1. 1 .

 1 Fig. S1.1. UV-Visible spectra of MnTPP-1CP and MnTPP-1CP/ CRGO measured in DMF.

Fig. S1. 2 .

 2 Fig. S1.2. FT-IR of (a) MnTPP-1CP and (b) MnTPP-1CP/C`RGO

Fig. S1. 3 .

 3 Fig. S1.3. Relationship of ΔEp and ψ versus v -1/2 in benzoquinone and Fe(CN)6 3-/4-, from which the ks of CRGO, H2TPP-1CP/CRGO, and H2TPP-4CP/CRGO were calculated.

(scheme 2 . 1 )

 21 where each step has been characterized through different analytical techniques such as FT-IR, UV and electrochemical measurements. The immobilization of ssDNA and hybridization with complementary sequences lead to the change of electrochemical properties for MnTPP-1CP/CRGO. The condition for the process of DNA sensor construction and hybridization, of which the concentration ratio of MnTPP-1CP and CRGO, the concentration of activated reactants (EDC and NHS), and the hybridization time have been optimized by various electrochemical tests. EIS experiments demonstrate an increase of impedance after DNA probe grafting and hybridization. A detection limit of 6 × 10 -14 M has been calculated and a dynamic range of 1× 10 -16 M to 1× 10 -11 M has been shown. Selectivity of the DNA sensor has been investigated in presence of non-complementary and one-mismatched 121 Phosphate buffers saline (PBS) pH=7.4, contained 10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl and 137 mM NaCl was prepared with double distilled water, filtered by 0.22 µm membrane filter and stored at 4 º C until use. All DNA sequences were provided by Eurogentec company. The DNA probe was a 25bases sequence with six carbon chains as spacer and with amine group on 5' phosphoryl terminus: NH2-C6 -5'-TCA-ATC-TCG-GGA-ATC-TCA-ATG-TTA-G-3'. The sequence of 25-bases target specific for DNA probe was 5'-CTA-ACA-TTG-AGA-TTC-CCG-AGA-TTG-A-3'. The non-complementary and the one-mismatched 25-bases oligonucleotide was 5'-CTA-ACA-TTG-AGA-TTC-CGA-GAT-TGA-GAT-CTT-C-3' and 5'-CTA-ACA-TTG-ACA-TTC-CCG-AGA-TTG-A-3' respectively.

  123 reflectance (ATR) crystal of germanium.Electrochemical measurements were performed using an AUTOLAB PGSTAT 100 electrochemical analysis system controlled by NOVA software (Methrom). The three-electrode cell was purchased from BASI and consisted of glassy carbon electron (GCE) (surface 0.071 cm 2 ) as working electrode, a platinum as counter-electrode and Ag/AgCl as reference electrode. The analyses were performed by cyclic voltammetry (CV), square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS).CV analysis were realized in 0.1 M KCl with 5 mM [Fe(CN)6] 3-/4-species in the range of potential from -0.2 to -0.6 V with scan rate 50 mV.s -1 .SWV measurements were performed in PBS in the range of potential of 0.6 to -1.0 V with potential amplitude of 50 mV and step height of 10 mV frequency of 1.25 Hz, the measurement is corresponding to scan rate of 12.5mV/s. EIS analysis were performed in [Fe(CN)6] 3-/4-at 0.15 V vs. Ag/AgCl at DC potential of 10 mV with the frequency ranging from 100 KHz to 0.1 Hz. The impedance measurements in PBS solution were obtained at -0.3 V vs. Ag/AgCl at DC potential of 10 mV with the frequency ranging from 100 KHz to 0.01 Hz.

1 . 2 . 1 .

 121 Scheme 2.1. Schematic representation of DNA sensor

  DNA sensors were activated by immersing the MnTPP-1CP/CRGO electrode in PBS solution including 20 mM EDC in presence of 35 mM NHS or without NHS. The electrochemical detection was monitored by SWV. The estimated relative current (ΔI/I0) was shown in Fig. 2.2A. The addition of the NHS has positive effect on the performance of the DNA sensor. Indeed, the presence of NHS as activated reactant increases considerably the relative variation of current peak of the porphyrin complex.The effect of EDC concentration has also been investigated in the condition of the fixed ratio of EDC and NHS (1:1.75). The result exhibits that the higher concentration of EDC has negative effect on the performance of DNA senor. The optimized activated condition for activating carboxylic group should to be 20 mM EDC and 35 mM NHS.

2. 3 )Fig. 2 . 3 .

 323 Fig. 2.3. EIS obtained after DNA detection of 1fM of target after different hybridization times(20, 30, 40, 50 and 60 min.) in 5 mM [Fe(CN)6] 3-/4-; Insert: calibration curve.

Fig. 2 .Fig. 2 . 4 .Fig 2 . 5 .

 22425 Fig. 2.4B shows the EIS measurements performed in 5 mM [Fe(CN)6] 3-/4-species after the modification of GCE surface. Results demonstrate an obvious decrease of electron transfer resistance (Rct) after the interaction of ssDNA and MnTPP-1CP (Fig. 2.4B, b). The increased Rct verifies the successful covalently interaction between ssDNA and MnTPP-1CP. The reason for this variation can be ascribed to the blocking of ions diffusion to the electrode surface. The stability over the time of the electrochemical signal of the metalloporphyrin was evaluated by SWV experiments in PBS (Fig. S2.2). Scarce significant variation of both oxidation and reduction of the current and potential is observed, demonstrating for the first time the excellent stability of the biolayer due to strong interactions between MnTPP-1CP and CRGO through π-π stacking.

Fig 2 . 7 .

 27 Fig 2.7. Histogram of Rct/ Rct,ssDNA for DNA sensor after detection of 1nM of cDNA, cDNA with one-base mismatched, and non-complementary sequence, respectively.

Fig. S2. 1 .Fig. S2. 2 .

 12 Fig. S2.1. FT-IR spectra of MnTPP-1CP/CRGO and after covalently interactedwith ssDNA through amide link.

1 .

 1 The hybridization of c PCR samples was performed by immersing the electrode modified DNA probe specific for PCR samples in solution of 1 fM c PCR for 40 min at 47 º C. The same conditions were applied for the detection of the same concentration of nc PCR.

1

 1 Construction of DNA sensor DNA sensor construction involves covalent attachment of single stranded DNA probes (ssDNA) labelled with an amine group with 6 carbon atoms as spacer (C6 amino link). The ssDNA probe was covalently bonded to the carboxylic groups present in H2TPP-1CP/CRGO hybrid complex through an amide link. The reaction was performed in presence of EDC and NHS, as activating agents for one hour. After washing the modified electrode with distilled water to remove any reactants left. The formation of DNA sensor was monitoring by electrochemical impedance measurement (EIS) and cyclic voltammetry (CV) in both [Fe(CN)6] 3-/4-and pbenzoquinone solution and by square wave voltammetry (SWV) in p-benzoquinone solution only. Impedance measurement (EIS) was used to characterize electrochemical properties of the modified layer, especially the interface between the modified electrode and the solution, including the impedance of the layer, capacitance of the electric double layer and the electron transfer resistance (Rct). The results performed in PBS before (Fig 3.1, curve a) and after the reaction of ssDNA probe with H2TPP-1CP (Fig. 3.1, curve b) are shown in Fig. 3.1 and data in supplementary information (Table

PART B : Fig. 3 . 1 .

 :31 Fig. 3.1. Nyquists plots measured in PBS solution with modified electrode H2TPP-1CP/CRGO (a) and ssDNA/H2TPP-1CP/CRGO (b).

Fig. 3 . 2 .Schema 3 . 1 .

 3231 Fig.3.2. (A) Cyclic voltammograms and (B) Nyquists plots of H2TPP-1CP/CRGO before (solid black line or a) and after (dashed black line or b) immobilization of ssDNA. Recorded in 0.1 M KCl with 5 mM [Fe(CN)6] 3-/4-species

Fig. 3 .

 3 Fig.3.3 shows the SWVs of the modified electrode surface performed in benzoquinone solution before and after ssDNA immobilization. The voltammograms exhibit a decrease of reductive current of benzoquinone to hydroquinone after ssDNA covalently interacted with H2TPP-1CP/CRGO platform. This can be explained by either to the low electron transfer or to the slow diffusion of ions to the electrode surface during redox process, due to the attachment of ssDNA molecules, as observed in previous work2,3 . The electrochemically reduced signal current decreases in proportional to the amount of the ssDNA immobilized on the H2TPP-1CP/CRGO layer. On the basis of the variation of the charge Q obtained under the cathodic wave

Fig. 3 . 3 .

 33 Fig. 3.3. SWVs of H2TPP-1CP/CRGO before (solid line) and after (dashed line) immobilization of ssDNA, recorded in 0.1 M KCl with 1 mM , scan rate of 12.5mV s -1 .

Fig. 3 . 4 .-

 34 Fig. 3.4. Detection of complementary target of different concentrations: 10 aM; 1 fM; 10 fM; 100 fM; 1 pM; 10 pM; 1 nM; 10 nM. in 5 mM [Fe(CN)6] 3-/4-, (A) EIS measurments, the inset is the relative changes of the electron transfer resistance ΔRct/Rct ssDNA vs. concentration (B) CV.

Fig. 3 .Fig. 3 . 6 .

 336 Fig. 3.6B shows a 30% variation of the current in presence only of 1 fM of nc DNA. This positive signal of the sensor can be explained by undesired interactions of nc DNA with the modified electrode surface, such as Van der Waals interactions, stacking interactions with aromatic DNA bases. Similar results were also observed during the detection of nc DNA of the DNA sensor based on MnTPP/CRGO materials (Part B. chapter Ⅱ).To eliminate this phenomenon, we proposed to increase the number of negative charges of the nanomaterials on the electrode surface. Indeed, with an electrode surface charged more negatively, we expected that the DNA strands, also charged negatively, less interact. A solution is to increase the number of carboxylic functions on the porphyrins skeleton that becomes negatives at pH 7. Thus we proposed to synthesize a new porphyrin bearing four carboxylic groups on phenyl groups (H2TPP-4CP) and to integrate in the DNA sensor

Fig. 3 . 7 .

 37 Fig. 3.7. Nyquists plots measured in PBS solution (a) H2TPP-4CP/CRGO and (b) ssDNA/H2TPP-4CP/CRGO. A study of the ssDNA/H2TPP-4CP/CRGO surface, was performed by the CV and EIS in 5 mM negatively [Fe(CN)6] 3-/4-species after the immobilization of ssDNA (Fig. 3.8). CV experiments (Fig. 3.8A) show that after the immobilization of ssDNA, redox signal of [Fe(CN)6] 3-/4-species becomes more reversible (increase of the current intensity and E closed to 60 mV). The estimated electron resistance transfer (Rct) sharply decreases from 8.4 to 2.3 kΩ (the data is shown in Table.S3.5) (Fig. 3.8B).

  S3.5) (Fig.3.8B).

Fig. 3 . 8 .

 38 Fig. 3.8. (A) Cyclic voltammograms of immobilization of ssDNA and (B) Nyquists plots recorded in 0.1 M KCl with 5 mM [Fe(CN)6] 3-/4-of H2TPP-4CP/CRGO before (solid black line or a) and after (dashed black line or b).

Fig. 3 . 9 .

 39 Fig.3.9. SWVs of H2TPP-4CP/CRGO before (solid line) and after (dashed line) immobilization of ssDNA, recorded in 1 mM benzoquinone scan rate of 12.5mV s -1 .

Schema 3 . 2 . 2 . 2 Fig. 3 . 10 .

 3222310 Fig.10A). The regression equation is ΔRct/ Rct,ssDNA = 0.1425 lgCtarget +2.55 with regression coefficient of 0.99. The detection limit was estimated to be 6 × 10 -17 M (signal-to-noise ratio of 3 independent measurements, taking into accord the standard deviation of 0.2617).

Fig. 3 .Fig. 3 . 11 .Fig. 3 . 2 (

 331132 Fig. 3.11. SWVs experiments for detection in benzoquinone solution with the scan rate 12.5 mV.s -1

Fig. 3 .

 3 Fig. 3.12. (A) Plot of the relative changes of the peak current versus concentration of DNA targets on the H2TPP-4CP/CRGO in benzoquinone. (B) Histogram comparing hybridization signal intensities. Data points are the mean values ± RSD obtained with three measurements.

Fig. 3 . 13 .

 313 Fig. 3.13. AFM images of (a) ssDNA/H2TPP-1CP/CRGO and (b) ssDNA/H2TPP-4CP/CRGO; and (c), (d) line cross-section analysis of the AFM images respectively.

3. 3

 3 Summary and Comparison of Detection of ssDNA/H2TPP/CRGO The performance of the two DNA sensors, monitored by different electrochemical techniques (EIS and SWV) in presence of the two redox markers PART B: Results and discussion Chapter Ⅲ: DNA sensor based on H2TPP-nCP/CRGO 165 (Fe(CN)6 3-/4-and benzoquinone) are summarized in the Table 3.4, for better view.

Fig. 3 . 15 .

 315 Fig. 3.15. Histogram comparing hybridization signal intensities for DNA sensor after detection of 1 fM of c DNA and nc DNA sample. Data points are the mean values ± RSD obtained with three independent experiments.

3 . 4 DNA-Fig. 3 . 15 .

 34315 Fig. 3.15. Detection of c PCR and nc PCR sequence in benzoquinone (A) SWVs measurements scan rate 12.5 mV.s -1 (B) Histogram comparing hybridization signal intensities for detection of 1 fM of c PCR and nc PCR sample. Data points are the mean values ± RSD obtained with three independent experiments.

PART B :

 : Results and discussionChapter Ⅲ: DNA sensor based on H2TPP-nCP/CRGO 170 Supporting Information

  -Dimethylformaldehyde (DMF), acetonitrile (ACN), and β-phenylethylamine (β-PEA) were provided by Sigma-Aldrich, N-hydroxysuccinimide (NHS) from Fluka (Switzerland) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) from Sigma-Aldrich. Phosphate buffers saline (PBS) pH=7.4, contained 10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl and 137 mM NaCl was prepared with double distilled water, filtered by 0.22 µm membrane filter and stored at 4 º C until use. All DNA sequences were provided by Eurogentec company. The DNA probe was a 25bases sequence with six carbon chains as spacer and with amine group on 5' phosphoryl terminus: NH2-C6 -5'-TCA-ATC-TCG-GGA-ATC-TCA-ATG-TTA-G-3'. The sequence of 25-bases target specific for DNA probe was 5'-CTA-ACA-TTG-AGA-TTC-CCG-AGA-TTG-A-3'. The non-complementary and the one-mismatched 25-bases oligonucleotide was 5'-CTA-ACA-TTG-AGA-TTC-CGA-GAT-TGA-GAT-CTT-C-3' and 5'-CTA-ACA-TTG-ACA-TTC-CCG-AGA-TTG-A-3' respectively.

Fig. 4 .Fig. 4 . 1 .Scheme 4 . 1 .

 44141 Fig. 4.1 exhibits the CV of electrodeposition process after each cycles in presence of the three different concentrations of -PEA. In the three cases, the shape of voltammograms is similar. During the first cycle, a single broad and chemically oxidative irreversible peak at around +1.25V with a maximal intensity current (10 µA , 20 µA and 40 µA respectively) is observed and after each cycle the current decrease more or less rapidly.

4CP/CRGO/ 1 12 VFig. 4 . 3 .

 11243 Fig. 4.3. Nyquists plots measured in PBS solution with Dc potential of 10mV at frequency range of 100 KHz to 0.1 Hz with various modified electrode H2TPP-4CP/CRGO, H2TPP-4CP/CRGO/1β-PEA, and H2TPP-4CP/CRGO/2β-PEA

Fig. 4 . 4 .

 44 Fig. 4.4. AFM images of 1 β-PEA /GCE (a) and 2 β-PEA/GCE (c), and of H2TPP-4CP/CRGO/1 β-PEA (b) and H2TPP-4CP/CRGO/2 β-PEA (d), respectively.

4 .Scheme 4 . 2 .

 442 Scheme 4.2. The schematic graph for the orientation transform from perpendicular to parallel to the GCE surface.

190 3 .Fig. 4 . 5 .

 345 Fig. 4.5. SWVs of H2TPP-4CP/CRGO/1 β-PEA before (solid line) and after (dashed line) immobilization of ssDNA, scan rate of 12.5mV s -1 .

  SWV results exhibit different behavior of DNA sensing depending on the concentration of immobilized PEA. Indeed, the current intensity of benzoquinone decreases with the increasing concentration of cDNA targets for sensor based on H2TPP-4CP/CRGO/1β-PEA (Fig. 4.6 b) which presents the same behavior of the surface free of PEA. The formation of double-strand DNA structure blocks the surface and prevents diffusion process of p-benzoquinone species to it. However, sensors formed with with more concentrated -PEA layer on the electrode (Fig. 4.6 b and c), show response totally inversed where current intensity of benzoquinone increases with the increasing concentration of cDNA targets. The formation of the double-strand DNA structure during the hybridization releases the conductive surface that means the surface is less blocked favoring the reduction process of benzoquinone.The ssDNA lies certainly on the modified surface through interactions and in presence of complementary ssDNA the double-strand DNA structure goes up, releasing the surface. The ordered organization of the sensor layer afforded a "signal on" during the detection, that is, could be explained by high organization of the composite H2TPP-194 4CP/CRGO and high density of SS DNA on the surface which cover the graphene surface. When hybridization is performed double strand DNA are formed which is pick up from the surface leading to higher diffusion of redox probe and increase in current density. This interpretation should be confirmed in future by AFM image and other measurement such as EIS.

Fig. 4 .Fig. 4 . 7 .Fig. 4 . 8 .Fig. S4. 1 .Fig. S4. 2 .

 4474812 Fig. 4.7 shows the results of SWV analysis obtained after incubation of the DNA sensors based on H2TPP-4CP/CRGO/1 β-PEA, H2TPP-4CP/CRGO/2 β-PEA, and H2TPP-4CP/CRGO/4 β-PEA, respectively in successive concentrations of nc DNA targets ranging from 1 aM to 1 nM. These results clearly indicate that reduction current of benzoquinone is sensitively no affected for sensor based on high amount of β-PEA immobilized on the GCE surface (Fig. 4.7.b and c). For 1 fM nc DNA targets

4 )

 4 ABBREVIATIONS

Table of contents

 of .

  [START_REF] Zeng | Gold nanoparticles/water-soluble carbon nanotubes/aromatic diamine polymer composite films for highly sensitive detection of cellobiose dehydrogenase gene[END_REF][START_REF] Carpini | Oligonucleotide-modified screen-printed gold electrodes for enzymeamplified sensing of nucleic acids[END_REF]

Table 1

 1 DNA sensors developed with different indicators or intercalations

	Indicators	Method	Range	Sensitivity	Reference
	/intercalators
	[Fe(CN)6] 3-/4-	EIS	1.0 fM to 10.0 pM	0.3 fM	Bollo et al. 36
	[Fe(CN)6] 3-/4-	EIS	2×10 -12 to 2×10 -6 M	3.2×10 -13 M	Bo et al. 40
	[Fe(CN)6] 3-/4-	EIS	1×10 -11 to 1×10 -14 M	5.4×10 -15 M	Radhakrishnan et al. 41
	daunomycin	DPV	1.0×10 -12 to 1.0×10 -7 M 1.3×10 -13 M	Zhu et al. 38
	MB	DPV	1.0×10 -13 to 1.0×10 -8 M 3.2×10 -15 M	Huang et al. 42
	[Ru(NH3)6] 3+	DPV	1×10 -10 to 1.0×10 -7 M	3.5×10 -11 M	Wang et al. 43
	[Ru(bpy)3] 2+	CV	180 pM to 90 nM	90 pM	Yan et al. 44

Table 2

 2 DNA sensors based on CP or CPC*

	No Matrix	Range	Sensitivity Reference
	1	Polypyrrole	3.7 to 370 nM	1 nM	Peng et al. 49
	2	polyaniline	100 fg.mL -1 and 1 ng.mL -	100 fg.mL -1 Davis et al. 50
			1
	3	Boron doped diamond	10 -19 g.L -1 to 1 ng.L -1	10 -19 g.L-1 Weng et al. 51
		modified
		polyethylimine
	4	Poly 5-carboxyindole	1.0×10 -8 to 1.0×10 -18 M	1.0×10 -17 M Mohan	et
					al. 52
	5	Polyaniline/polyacylate	2.0×10 -7 to 5.0×10 -8 M	2.0×10 -8 M	Gu et al. 53
		modified Boron doped
		diamond
	*CPC: conducting polymer composites consist of one or more non-conducting polymers and conductive-filler
	materials distributed throughout the polymer matrix.

Table 3

 3 DNA sensors based on Polymer-nanocomposites (PNCs) modified electrode

	polymer combined with	Analyte				LOD	Reference
	nanoparticles
	PANI-chitosan	Hydroquinone			9.7×10 -7 M	Tang et al. 58
	PANI-AuNPs	DNA sequence associated	150 pM to 1 μM Spain et al. 26
		with S. aureus
	PEDOT-AuNP	DNA sequence associated	150 pM to 1 μM Spain et al. 59
		with S. aureus
	PPy-PANI-AuNPs	15	-	mer	DNA	1.0×10 -13 M	Wilson et al. 60
		oligonucleotides
	PPy-PEDOT-AgNPs	27	-	mer	DNA	5.4×10 -15 M	Radhakrishnan et al. 41
		oligonucleotides
	PPy-AuNPs	Designed target DNA from	8.4×10 -13 M	Nowicka et al. 61
		Lactococcus gene

Table 4

 4 Important parameters of carbon materials of different dimensionalities[START_REF] Chen | Progress in Materials Science[END_REF] .

	Isomer	Graphene	Fullerene	Carbon nanotube
	Dimension	2D	0D	1D
	Hybridization	sp 2	sp 2	sp 2
	Density (g/cm 3 )	2.26	1.72	1.2-2.0
	Bond length (Å)	1.42 (C=C)	1.40 (C=C)	1.44 (C=C)
			1.46 (C-C)
	Electronic	Zero-gap	Semiconductor	Meta/semiconductor
	properties	Semiconductor	Eg=1.9 eV	Eg=~0.3-1.1eV
		(~ 7200 S.m -1 )
	theoretical	(~ 2630 m 2 g -1 )
	specific surface
	area
	thickness of	0.35 to 1.00
	graphene	nm

  oxide was purchased from Aldrich. High purity graphite rods (99.995%, 6 mm diameter, 150 mm length) were purchased from Sigma-Aldrich. Sodium hydroxide (NaOH) was provided by Alfa Aesar. Hydrogen peroxide (H2O2) was purchased by Acros Organics.

Aqueous solutions were purified with Milli-Q water (18.2 MΩ cm). Pyrrole and dichloromethane (DCM) were distilled under argon prior to use. All other solvents were used without purification. Reactions were monitored by thin layer chromatography (TLC) performed on silica gel sheets containing UV fluorescent indicator (60 F254 Merck). Yields refer to chromatographically pure compounds. N, N-Dimethylformamide (DMF) was provided by Sigma-Aldrich. Phosphate buffers saline (PBS) pH=7.4, contained 10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl and 137 mM NaCl was prepared with double distilled water, filtered by 0.22 µm membrane filter and stored at 4 º C until use.

  Three different types of graphene nano-sheets were synthesized and compared. The first approach was explored by the electrochemical exfoliation of graphite and the others are

		O	H	H 3 CO			OCH 3
	H N	+	i	N	N H N H	N
			OCH 3
				H 3 CO		6	OCH 3
	Scheme 1.5. Synthesis of 5, 10, 15, 20-(p-methoxyphenyl)triphenylporphyrin (i)
		Propionic acid, 140 ℃, reflux 1.5
	3.2 Synthesis of Different types of graphene nano-sheets
	COOH 9 Scheme 1.4. Synthesis of H2TPP-4CP (9) (i) BBr3, DCM, rt, 18h, 86%; (ii) CsCO3, 6 iii 5-Bromovaleronitrile DMF, 60°C, 24h, 34%; (iii) Dioxane, HCl 12M, 80°C, 12h, 32% 7 81

reductive conversions from graphene oxide (GO) via either an electrochemical method to form ERGO nanomaterial or chemical reduced graphene oxide, denoted as CRGO. The electrochemical performance was optimized by cyclic voltammetry test in [Fe(CN)6] 3-/4-. The characteristics and electrochemical properties were analyzed by XPS, Raman, and CV.

Table 1 . 1 .

 11 Physical parameters extracted from XPS spectra.

	Peak Table : GO
	Name Peak BE FWHM	Area (P) CPS.eV Area (N)	Atomic %
			eV
	O1s	533.13 2.96	1180555.15	1180555.15	55
	C1s	286.66 4.27	980100.11	980100.11	45
	Peak Table :
	CRGO
	Name Peak BE FWHM	Area (P) CPS.eV Area (N)	Atomic %
			eV
	O1s	532.27 3.86	406502.21	2261.28	14
	C1s	285	3.07	1004332.75	14256.22	86

Table S1 .

 S1 1.

						C=O
							C=C
	H 2 TPP-4CP
	H 2 TPP-4CP/CRGO			C=O 1731cm -1	C=C 1602cm -1
			O-H			C=O 1711cm -1
		(A)					C=C
	4000	CRGO 3500	3000	2500	2000	1500
	Transmitance (%)	H 2 TPP-1CP/CRGO H 2 TPP-1CP		1731 cm -1 C=O	1582 cm -1 C=C C=C 1611 cm -1
			O-H 3256 cm -1		C=O 1727 cm -1	C=C
							1577 cm -1
	4000	3500	3000	2500	2000	1500
				Wavenumber (cm -1 )
				96

Table 1

 1 .1.

		0.06	(A)								0.06	(B)
		0.04	H 2 TPP-1CP/CRGO						0.04	H 2 TPP-4CP/CRGO
	Current (mA)	-0.02 0.00 0.02					50 mV/s		(mA) Current	0.02 -0.02 0.00				50 mV/s
		-0.04									-0.04
		-0.06					1000 mV/s			-0.06				1200 mV/s
			-0.4	-0.3	-0.2	-0.1	0.0	0.1	0.2			-0.4	-0.3	-0.2	-0.1	0.0	0.1	0.2
					Potential v.s. Ag/AgCl (V)						Potential v.s. Ag/AgCl (V)
							0.06
							(C)			H 2 TPP-1CP/CRGO
							0.04
							0.02				H 2 TPP-4CP/CRGO
						(mA)	0.00
						Current	-0.02
							-0.04
							-0.06
							5	10	15	20	25	30		35
									v	1/2 (mV s -1 )

Table 1 .

 1

	1. Electrochemical parameters obtained for electrodes of various
	modifications

Table S1 .2. Summary

 S1

	methoxyphenyl) tetraphenylporphyrin (6) (400 mg, 54 mmol) dissolved in 80 mL CH2Cl2 under argon. The crude solution was mixed during 18 hours at room temperature. The solution was then basified with a saturated Na2CO3 solution and washed with water until the neutrality. The organic phase was then dried over Na2SO4 and concentrated to give 319 mg (86%) of the crude product (7). Compound 7 was directly used in the next step without any purification. 1 H NMR (250MHz, CDCl3): 8.88 (m, 8 H, pyrrol-Hmeso), 8.24 (m, 6 H, Ar-H), 8.07 (d, 3 JH,H = 8.5 Hz, 2H, Ar-H), 7.80 (m, 9H, Ar-H), 7.12 (d, 3 JH,H = 8.25 Hz, 2H, Ar-H), -2.06 (ls, 2H, NH). 5,10,15,20-[4-(valeronitrile)phenyl] tetraphenylporphyrin (8) On a solution of porphyrin (7) (50 mg, 0.073 mmol, 1eq) in DMF (25 mL) was added 0.48g Cs2CO3 (1.46 mmol, 20eq) under argon for stirring 2h at room temperature. H NMR (300MHz, CDCl3):  8.87 [8H, d, J=7.8 Hz], 8.24 [6H, d, J=6.9 Hz], 8.14 [2H, d, J=8.4 Hz], 7.77 [9H, t, J=6.6 Hz, Ha], 7.28 [2H, d, J=6 Hz], 4.3 [2H, t, J=5.55Hz], 2.6 [2H, t, J=6.75Hz], 2.19 [4H, m],-2.74 [2H, s];. FT-IR (ATR) 2093 cm -1 (CN). 5,10,15,20-[4-(carboxylbutyloxy)phenyl] tetraphenylporphyrin (9) Nitrile was hydrolyzed by HCl 12M and afforded carboxylic porphyrin 9. On a solution of porphyrin (8) (100 mg, 0.1 mmol) in 4 mL dioxanne, was added 1.8 mL of a concentrated HCl solution. The reaction was running vigorously at 80°C during 12 hours. Dichloromethane was added and the crude mixture was washed with water, dried over Na2SO4. Crude product was obtained with 32% of yield (35 mg) as a pure Chapter I: Synthesis and properties of nanomaterials 1CP GO ⅴ O-H 3248 ⅴ O-H 3259 ⅴ O-H 3256 ⅴ C= O 1724 ⅴ C=O 1729 ⅴ C=O 1727 ⅴ C= C 1583 ⅴ C=C 1611 ⅴ C=C 1572 1466 1462 1456 ⅴ C-OH 1224 ⅴ C-OH 1252 ⅴ C-O-C 1074 ⅴas CH2 2921 ⅴas CH2 2955 ⅴas CH2 2927 ⅴs CH2 2854 ⅴs CH2 2844 ⅴs CH2 2844 2352 2341 2385 ⅴ C=C in pyridy l aromat ic ring 1511 1661 1152 1213 δ N-H 964 δ N-H 960 CRG O Bon d Wavenum ber H2TP P-4CP Bond Wavenum ber H2TPP-4CP/CR GO Bond Wavenum ber ⅴ O-3248 ⅴ O-H 3259 ⅴ O-H 3253 CH2 CH2 CH2 ⅴs CH2 2854 ⅴs CH2 2880 ⅴs CH2 2888 2822 2352 2352 2348 1771 ⅴ C=C in pyridy l aromat ic ring 1506 ⅴ C=C in pyridy l aromat ic ring 1501 1412 ⅴ C=N 1356 ⅴ C=N 1354 1168 1138 1064 δ N-H 953 δ N-H 975 Rs (Ω) Rct1 (KΩ) CPE1 (μMho) N1 W1 (μMho) Rct2 (KΩ) CPE2 (μMho) N2 CRGO 92.4 ±1.2 135 ±1.8 18.7±1.2 0.94 ±0.6 166±2.57 0.07 H2TPP-1CP/RGO 82.8± 1.5 72.6 ±5.2 28.0± 2.9 0.96 ±1.5 59.4 ±2.3 99.3± 9.5 0.53 ±2.1 0.06 H2TPP-4CP/RGO 91.6±1.1 102± 3.9 20.3± 2.2 0.98 ±1.1 72.4 ±2.2 98.9± 10.2 0.56 ±2.1 0.05 PART B: Results and discussion Chapter I: Synthesis and properties of nanomaterials 400 450 500 550 600 650 700 0,0 Wavenumber (nm) powder. PART B: Results and discussion C= 2.0 [4H, m]; -2.7 [2H, s, NH]. HRMS (APCI MS) m/z calcd. for C64H62N4O12, (M+H) + 1079.22, found 1079.22. UV-vis spectrum in DMF: max, 419 nm; 515 nm; O ⅴ 1583 ⅴ C=C 1605 ⅴ C=C 2,4 465 1578 MnTPP 2,0 MnTPP/RGO C= 551 nm; 592 nm; 647 nm. Table S1.1. Peak assignment for CRGO, H2TPP-1CP, H2TPP-4CP, H2TPP-1CP/CRGO, and H2TPP-4CP/CRGO obtained from FTIR spectra CRG O Bon d Wavenum ber H2TP P-Bond Wavenum ber H2TPP-1CP/CR Bond Wavenum ber C 1466 1462 1,6 1406 ⅴ C-OH 1224 ⅴ C-OH 1246 ⅴ C-OH 470 1257 ⅴ C-ⅴas 2921 ⅴas 2955 ⅴas 2955 0,4 O-C 1074 0,8 1,2 Absorbance
	H
	ⅴ
	110 111 112 113

5,10,15,20-(4-hydroxyphenyl) tetraphenylporphyrin

(6)

Boron tribromide (1.16mL, 22 eq.) was added at 0°C onto 5,10,15,20-(4-And then 0.17 ml 5-bromopentanenitrile was added (1.46 mmol, 20eq) into the solution and the mixture was heated at 60°C during 2 days. The solution was concentrated under vacuum and extracted with CH2Cl2 and washed with H2O. The organic phase was then dried over Na2SO4 and concentrated to give 25 mg (34%) of the crude product (8) and was directly used for the next step.

1

1 H NMR (300MHz, CDCl3):  8.9 [8H, m]; 8.3 [6H, m]; 8.2 [2H, d, J= 8.1 Hz]; 7.8 [9H, m]; 7.2 [2H, d, J= 7.5Hz]; 4.2 [2H, t, J=4.8 Hz]; 2.6 [2H, t, J= 6.9Hz]; of estimated EIS parameters obtained in PBS buffer, pH 7.4. Results are mean ±S.D. obtained from 3 independent measurements in each series

Table S1 . 3 .

 S13 Electricals parameters measured from the fitting data of the Nyquist plot

	MnTPP-	89.9	894 ±1.5 1.48±1	0.86				0.0
	1CP	±1.4		.2		±0.24				9
	MnTPP-	64.2	132 ±3.0 22.8±1	0.86			144	11.3±1	0.68±2	0.1
	1CP/CRG	±2.9		.7		±1.1			±5 .9	1.4	.0	6
	O
			0.075
			0.070
			0.065
		Ep (V)	0.060
			0.055
			0.050
			0.045
			0.040
			1.0	1.2	1.4	1.6	1.8	2.0	2.2	2.4
						v -1/2 (mV s -1 )
		Rs	Ret1	CPE1	N1		W1	Ret2	CPE2	N1	χ²
		(Ω)	(KΩ)	(μMh			(μMh	(KΩ)	(μMh
				o)			o)		o)
	CRGO	92.4 ±1.2	135 ±1.8 18.7±1 .2	0.94 ±0.6 0.0 114 166±2. 57

  Plot of the relative changes of the electron transfer resistance ΔRct/ Rct ss DNA versus concentration. Data points are the mean values ± RSD obtained with four independent experiments.

		1.6	R/R 0 =0.1878 lgC target +3.428
		1.4	R=0.9935
		1.2
	R/R 0	0.8 1.0
		0.6
		0.4
		0.2
		1E-17	1E-15	1E-13	1E-11	1E-9	1E-7
					C target /M
	Fig 2.6.
	2.6.
						131

The ΔRct corresponds to the difference between Rct value of the ssDNA immobilized on the electrode surface and that after hybridization with cDNA (ΔRct =Rct,dsDNA -Rct,ssDNA). The regression equation is ΔRct/ Rct,ssDNA = 0.188 lg Ctarget +3.428 with regression coefficient of 0.993. The detection limit is estimated to be 2×10

-16

M (signal-to-noise ratio of 4 independent measurements, taking into accord the standard deviation of 0.043). The DNA sensor based on MnTPP-1CP/CRGO nanomaterial exhibits a high sensitivity probably for all previous ascribed reasons such as the high specific surface of CRGO and the short distance between MnTPP-1CP and CRGO which increases the electron transfer. Comparing with other nanomaterials based on CRGO reported in literature for the DNA detection, our work presents an excellent sensing platform (Table

2

.1).

Table 2 . 1 .

 21 Comparison of the proposed method with some reported DNA sensors

	Detection	Detection	indicator	Ref
	technique	limit (M)
	EIS	1.0 × 10 13	Label-free ERGO-modified electrode	[ 4 ]
	EIS	1.1 × 10 13	GO anchored on a diazonium-functionalized	[ 5 ]
			electrode
	SERS a	1.0 × 10 11	Graphene CVD associated with gold nanoparticles	[ 6 ]

Table 3 .

 3

	2. Summary of the diffusion coefficient (D0) of the redox marker
	[Fe(CN)6] 3-/4-and p-benzoquinone for H2TPP-4CP/CRGO and ssDNA/H2TPP-
	4CP/CRGO

  3-/4-], compared to slightly increase relative Rct of H2TPP-1CP/CRGO (+ 5%) after the immobilization of ssDNA, the Rct/Rct decreased CRGO nanomaterial become more conductive after grafting ssDNA on it that can be explained by a repartition of charges differently with such material.

	PART B: Results and discussion	Chapter Ⅲ: DNA sensor based on H2TPP-nCP/CRGO
	obviously with a relative value of -73 % for H2TPP-4CP/CRGO. This indicates that
	the H2TPP-4CP/
		163

Table 3 .

 3

	3. Summary of data for ssDNA/H2TPP-1CP/CRGO and ssDNA/H2TPP-4CP/CRGO
				In [Fe(CN)6] 3-/4-	In
							benzoquinone
		Γ	Thickness	ΔRct/Rct ΔD0/	Δks/	ΔD0/	Δks/ ks
		(nmol	(nm)		D0	ks	D0
		cm -2 )
	ssDNA/H2TPP-	2.2±0.65	30	5%	-23%	-	-50%	-57%
	1CP/CRGO					75%
	ssDNA/H2TPP-	4.6±0.43	6	-72.6% 60%	-	-	-
	4CP/CRGO					50%	32.1%	37.5%

Table 3 .4. Summary of detection data EIS in [Fe(CN)6] 3-/4- SWV in benzoquinone LOD (M) Linear range (M) LOD (M) Linear range (M) nc 1fM (ΔI/I0) H2TPP- 1CP/CRGO dsDNA

 3

		7×10 -14	10 -11 ~10 -9	2×10 -18	10 -18 ~10 -12	30%
	H2TPP-	6×10 -17	10 -17 ~10 -9	7×10 -19	10 -18 ~10 -11	15%
	4CP/CRGO
	dsDNA

Table S3 .

 S3

		1. Summary of estimated EIS parameters of H2TPP-1CP/RGO and
	ssDNA/H2TPP-1CP/CRGO obtained in PBS buffer, pH 7.4. Results are mean ±S.D.
		obtained from 3 independent measurements in each series
		Rs	Rct1	CPE	N1	Rct2	CPE	N2	Thick	Capacit	χ 2
		(Ω)		1		(KΩ)	2		ness	ance
			(KΩ)	(μM			(μM		(nm)	(μF)
				ho)			ho)
	H2TPP-	82.8	72.6	28.0	0.96	59.4	99.3	0.53±	72±9.	0.0027 0.
	1CP/CR	±1.5	±5.2	±2.9	±1.5	±2.3	±9.5	2.1	3		06
	GO
	ssDNA/H	87.0	66.9	48.2	1.1±	166±	41.7	0.575	31±3.	0.0064 0.
	2TPP-	±2.5	±1.5	±2.6	1.7	1.5	±3.4	±2.2	5		03
	1CP/CR
	GO

Table S3 .

 S3

	PART B: Results and discussion	Chapter Ⅲ: DNA sensor based on H2TPP-nCP/CRGO
				3			6	3	8
	1nM	104±0.6 6.52±3.	4.45±2.	0.83±0.4	320±7.	0.36±10.	0.03
				9			8	4	6
		0.070	ssDNA/H 2 TPP-1CP/CRGO in Benzoquinone	1.8
								1.6
		0.065						1.4
		0.060						1.2
	 Ep (V)	0.050 0.055						0.8 1.0	
		0.045						0.6
		0.040						0.4
		0.035						0.2
		1.0	1.5			2.0	2.5	3.0	3.5
							v -1/2 (mV s -1 )
		0.09					1.8
				ssDNA/H	TPP-4CP/CRGO in Benzoquinone
						2
								1.6
		0.08
		Rs		Rct			CPE1	N1	CPE2	N2 1.4	χ 2
		0.07
	H2TPP-	(Ω) 0.05 100±0.5 3.49±3.2 10.8±2.6 0.742±0.4 249±2.7 0.392±4.1 0.04 (KΩ) 1.2 (μMho) (μMho) 0.8 0.06  Ep (V) 1.0 
	1CP/CRGO							0.6
	ssDNA/H2TPP-	117±0.8 3.56±1.2 5.09±3.3 0.795±0.5 335±2.6 0.04	0.5	0.04
	1CP/CRGO							0.4
		0.03
								0.2
	1.0 Table S3.3. Summary of estimated EIS parameters of ssDNA/H2TPP-1CP/CRGO and 1.5 2.0 2.5 3.0 3.5 4.0 4.5 v -1/2 (mV s -1 )
	dsDNA/H2TPP-1CP/CRGO obtained in 0.1 M KCl containing 5 mM [Fe(CN)6] 3-/4-
	solution. Fig. S3.1. Relationship of ΔEp and ψ versus v -1/2 in benzoquinone, from which the ks
	Rs of ssDNA/H2TPP-1CP/CRGO, and ssDNA/H2TPP-4CP/CRGO were Rct1 CPE1 N1 CPE2 N2	χ 2
		(Ω)	(KΩ)	(μMho) calculated.	(μMho
								)
	ssDNA/H2TPP	95.6±0.6 2.65±2.	7.24±3.	0.778±0.5	370±2.	0.396±4.	0.02
	-1CP/CRGO			9			3	3	6	0
	10aM	100±0.5	3.29±2.	7.5±2.8 0.784±0.5 356±3.	0.399±4.	0.01
		5		9				0	6	8
	1fM	99.9±0.5 4.35±3	6.46±2.	0.798±0.4	340±4.	0.386±5.	0.01
							6	3	0	9	9
	100fM	94.5±0.6 5.49±2. 5.86±2. 0.808±0.4 408±4.	0.461±7	0.02
							171

2. Summary of estimated EIS parameters of H2TPP-1CP/RGO and ssDNA/H2TPP-1CP/CRGO obtained in 0.1 M KCl containing 5 mM [Fe(CN)6] 3-/4- solution.

Table S3 . 4 .

 S34 Summary of estimated EIS parameters of H2TPP-4CP/RGO and ssDNA/H2TPP-4CP/CRGO obtained in PBS buffer, pH7.4.

		Rs	Rct1	CPE	N1	W1	Rct2	CPE2	N	χ 2 Thick	Capacit
		(Ω)		1		(μM	(KΩ	(μMh	2		ness	ance
			(KΩ	(μM		ho)	)	o)			(nm)	(μF)
			)	ho)
	H2TPP-	91.6	102±	20.3±	0.98		72.4	98.9±	0.	0.	20.4±1	0.009
	4CP/CRGO	±1.1	3.9	2.2	±0.0		±2.2	10.2	56	05	.2
					1				±0
									.0
									1
	ssDNA/H2TP	102	116±	20.5±	1.03		154	28.8±	0.	0.	6.5±0.	0.029
	P-	±4.1	3.1	2.1	±0.0		±3.2	4.2	64	04	5
	4CP/CRGO				1				±0
									.0
									1

Table S3 .

 S3

	PART B: Results and discussion	Chapter Ⅲ: DNA sensor based on H2TPP-nCP/CRGO
	7 2.32±2. 7 H2TPP-4CP/CRGO/2 β-PEA platform afford less non-specific ssDNA adsorption 3 8 1 4 100aM 89.1±1. 2 15.1±4. 5 0.823±0.0 1 386±2. 8 0.475±0.0 3 0.03 with only 2.5% of current variation Materials and Methods
	1fM	96.8±1.	2.67±2.	13.1±4.	0.838±0.0	374±2.	0.476±0.0	0.03
	100fM	2 94.2±1. 1	5 3.31±2. 2 PART B: 3 8 11.4±3. 9 0.849±0.0 8	9 361±3. 2	2 0.482±0.0 3	0.04
	1nM	96.7±0.	3.87±1.	9.85±2.	0.868±0.0	415±2.	0.466±0.0	0.02
		8	4		6		5		8	4
		0	RESULTS
		-5
	-15 -10 AND DISCUSSION (A)
	Current	-25 -20
		-30
		-35	GCE				I/I=15.4%
		-40	Immerse in 1 nM Target
		-0.4	-0.3		-0.2	-0.1	0.0	0.1	0.2
	Rs (Ω) CHAPTER Ⅳ: Rct1 (KΩ) CPE1 (μMho) N1 CPE2 (μMho Potential v.s. Ag/AgCl (V) Fig. S3.2. SWVs of GCE (solid line) and after immersion in 1 nM DNA target (dashed N2 χ 2 ) H2TPP-4CP/CRGO 91.7±3. 5 8.43±5.1 13.3±2. 0 0.731±0.0 4 294±10 0.355±0.01 3 0.01 line), recorded in 0.1 M KCl with 1 mM benzoquinone in the range of potential from -0.4 to 0.2 V with the scan rate of 12.5mV s -1 . Self-assembled selective DNA
	ssDNA/H2TP P-4CP/CRGO	95±0.5 2.28±0.8 6	9.74±2. 2 sensor 0.74±0.03 395±1. 3	0.502±0.01 0.01 5
	Table S3.6. Summary of estimated EIS parameters of ssDNA/H2TPP-4CP/CRGO and
	dsDNA/H2TPP-4CP/CRGO obtained in 0.1 M KCl containing 5 mM [Fe(CN)6] 3-/4-
		solution at applied potentiel 0.15 V vs. Ag/AgCl.
		Rs	Rct1		CPE1	N1		CPE2	N2	χ 2
		(Ω)	(KΩ)	(μMho)			(μMho
									)
	ssDNA/H2TPP	96.8±0.	1.92±2.	16.2±2.	0.753±0.0	465±1.	0.481±0.0	0.00
	-4CP/CRGO	7	2		9		5		8	4	9
	10aM	101±0.9 1.82±2. 18.5±4. 0.809±0.0 390±2. 0.465±0.0 0.02
						173 175

5. Summary of estimated EIS parameters of H2TPP-4CP/RGO and

ssDNA/H2TPP-4CP/CRGO obtained in [Fe(CN)6] 3-/4-at 0.15 V vs. Ag/AgCl.

Table 4

 4

	.2). Compared

due to two-dimensional -stacking interactions with aromatic rings of -PEA molecules or carboxylic groups of porphyrins are more accessible for DNA attachment. Furthermore, values of ΔQ and so ΔΓ are in the same order for the three materials: H2TPP-4CP/CRGO/1 β-PEA, H2TPP-4CP/CRGO/2 β-PEA and H2TPP-4CP/CRGO/4 β-PEA that demonstrate a significant effect of the -PEA concentration for layer where optimum is obtained with layer formed with 2mM.

Table 4 .

 4

		2. Summary of calculated parameters after the immobilization of ssDNA for each
			nanomaterials
			ΔQ (mC)	ΔΓ (nmol cm -2 )
		ssDNA/H2TPP-	0.06	4.6
		4CP/CRGO
		ssDNA/H2TPP-	0.114	8.6
		4CP/CRGO/1 β-
		PEA
		ssDNA/H2TPP-	0.15	11.3
		4CP/CRGO/2 β-
		PEA
		ssDNA/H2TPP-	0.08	6.0
		4CP/CRGO/4 β-
		PEA
	4.	Detection of DNA target
		Hybridization reaction was performed with complementary targets (cDNA) by

1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-12 1E-11 1E-10 1E-9 1E-8 1E-7

		-2	(D)	dsDNA/H 2 TPP-4CP/CRGO/4 -PEA
		-4
		-6
	Current (A)	-10 -8 -12	(B)	dsDNA/H 2 TPP-4CP/CRGO/-PEA 0.30 0.0348 lgC(M)+0.7504 0.25 R=0.99 1aM
											I/I0	0.20
		-14										0.15
	(A)	-16			1nM						0.10 1E-19 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-12 1E-11 1E-10 1E-9 1E-8 Concentration of cDNA target (M)
	Current		-0.4	1 nM -0.3	0.40 0.45 0.0 0.04065 lgC(M)+0.7668 0.1 Potential (V) v.s. Ag/AgCl -0.2 -0.1	0.2
												0.35	R=0.99
												0.30
	0.20 0.25 Fig. 4.6. SWVs experiments for detection of cDNA targets (1 aM to 1 nM). scan rate I/I0
												0.15
	0.05 0.10 12.5 mV.s -1 1 mM benzoquinone. H2TPP-4CP/CRGO/1 β-PEA (A), H2TPP-
	0.00 4CP/CRGO/2 β-PEA (B), and H2TPP-4CP/CRGO/4 β-PEA (C). The insets: Concentration of cDNA target (M) 1 aM
	logarithmic relationship Data points are the mean values ± RSD obtained with three
						Potential (V) v.s. Ag/AgCl independent experiments.
		0
			(B)	dsDNA/H	2 TPP-4CP/CRGO/2-PEA
		-5
	(A)	-10
	Current	-20 -15			1aM						0.5
													  0.044	lgC(M)+0.8468
												0.4	R=0.99
		-25
												0.3
												I/I 0
												0.2
		-30
												0.1
					1	nM
		-35										0.0
												1E-19	1E-18	1E-17	1E-16	1E-15	1E-14	1E-13	1E-12	1E-11	1E-10	1E-9	1E-8
													Concentration	of	cDNA	target	(M)
		-40
			-0.4	-0.3		-0.2	-0.1		0.0	0.1	0.2
								Potential	(V)	v.s.	Ag/AgCl
								193

Table 4 . 3 .

 43 Summary of performance of DNA sensors

	LOD (M) Linear range
	(M)

ⅴ C=O

] 3-/4-

GENERAL INTRODUCTION PART B: Results and discussion

Chapter Ⅲ: DNA sensor based on H2TPP-nCP/CRGO 142 Electrochemical measurements were performed using an AUTOLAB PGSTAT 100 electrochemical analysis system controlled by NOVA software (Metrohm). The three-electrode cell was purchased from BASI and consisted of glassy carbon electron (GCE) (surface 0.071 cm 2 ) as working electrode, a platinum as counter-electrode and Ag/AgCl as reference electrode. The analyses were performed by cyclic voltammetry (CV), square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS).

CV analysis were realized in 0.1 M KCl with 5 mM [Fe(CN)6] 3-/4-species in the range of potential from -0.2 to 0.6 V at various scan rates in the range 0.005-1 V s -1 and also analyzed in 0.1 M KCl with 1 mM benzoquinone in the range of potential from -0.4 to 0.2 V at various scan rates in the range 0.05-1 V s -1 .

SWV measurements were performed in 0.1 M KCl with 1 mM benzoquinone in the range of potential from -0.4 to 0.6 V with potential amplitude of 50 mV and step height of 10 mV frequency of 1.25 Hz, the measurement is corresponding to scan rate of 12.5 mV/s. EIS analysis were performed in [Fe(CN)6] 3-/4-at 0.15 V vs. Ag/AgCl at DC potential of 10 mV with the frequency ranging from 100 KHz to 0.1 Hz. The impedance measurements in PBS solution were obtained at -0.3 V vs. Ag/AgCl at DC potential of 10 mV with the frequency ranging from 100 KHz to 0.01 Hz.

Construction of DNA sensor

A drop of 2 µL of the prepared H2TPP-1CP/CRGO suspension were deposited onto a glassy carbon electrode surface (GCE) followed by drying step under the lamp.

The modified surface was then dried and rinsed with distilled water to remove no specific adsorptions. Finally, the H2TPP-1CP/CRGO surface was activated with a freshly prepared 10 mM PBS solution (pH=7.4) containing 20 mM EDC, 32 mM NHS. The activation step was running for 1h at room temperature. Then the electrode was immersed in 1µM solution of ssDNA probe prepared in PBS buffer for 1h at

Fig. 4.2. SWVs of 1 β-PEA/GCE (A), 2 β-PEA/GCE (B), and 4 β-PEA/GCE (C) respectively before (solid line) and after (dashed line) immobilization of H2TPP-4CP,

in 1 mM benzoquinone in the scan rate of 12.5mV s -1 .

Moreover, in comparing to the three types of layer, the amount of charge (Q) evaluated from the reduction wave increases of a factor of 2 after composite immobilization (Table 4.1). These results demonstrated that the introduction of graphene on the -PEA improves the conductivity of the electrode surface, facilitating both the reduction reaction and increasing the amount of reduced benzoquinone species.

GENERAL CONCLUSIONS

GENERAL CONCLUSIONS

The The comprehensive exploration including the molecular characteristics and electronic properties of the interaction between H2TPP with different carboxylic groups and CRGO were performed through SEM, AFM, UV-visible, FT-IR spectroscopic studies, and EIS measurements. The anchoring of porphyrin moieties weakens the interaction between the CRGO sheets, which further improves the dispersion of CRGO and formation of nano-sheets of graphene and edge surface. The obtained apparent association constant between the H2TPPs and CRGO was calculated using the Benesi-Hildebrand expression to be 85.1 g -1 L and 64.4 g -1 L, respectively for H2TPP-1CP/CRGO and H2TPP-4CP/CRGO confirming the strong interaction between H2TPP and CRGO sheets. The thickness of porphyrin obtained from EIS was calculated to be 72 9.3 nm and 20.4 1.2 nm, respectively for H2TPP-1CP/CRGO and H2TPP-4CP/CRGO, confirming that for H2TPP-1CP/CRGO, the nano-sheet is formed and the higher value, compared to H2TPP-4CP/CRGO, exhibits some aggregated domains upwards, and for H2TPP-4CP/CRGO, the porphyrin is anchored at the edge of CRGO and meanwhile, forms hydrogen bond with carboxylic group of porphyrins.

In order to further investigate the charactristics and electrochemical properties of coverage of hybrid nanomaterial H2TPP-nCP/CRGO, a porphyrin of manganese was synthezised. The manganese (Mn) was chosen as metallic cation due to its redox properties and reversible signal. Charactersitics of the layer (average surface coverage, electron transfert resistance, ) was investigated thanks the electrochemical signal response of the Mn 3+ /Mn 2+ redox complex. All the results demonstrate strong  stacking interactions between the manganese porphyrin (MnTPP-1CP) and CRGO and the ability of CRGO to immobilize a large amount of MnTPP-1CP offering a stable sensing layer.

For the first time, DNA sensors based on MnTPP-1CP was elaborated, where one carboxylic group was covalently functionalized with ssDNA on the GC electrode surface. The incorporation of a redox metal inside the macrocycle ring of porphyrin allows the electrochemical characterization of the film. Optimized conditions for the process of construction of DNA sensor and hybridization were performed including the concentration ratio of MnTPP-1CP and CRGO, the concentration of activated reactants (EDC and NHS), and the hybridization time. A detection limit of 2 × 10 -16

M was estimated with a moderate selectivity demonstrating that the combined porphyrin-graphene framework exhibits an excellent conductivity and a good electron transfer which contributes to amplify the redox signal of metalloporphyrin.

Furthermore, it demonstrate to be a very good candidate for direct DNA sensor and are very promising in DNA sensors applications. Nethertheless, after hybridization with cDNA with one-base mismatched and non-complementary sequences, the Rct/