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Surface enhanced resonance Raman scattering (SERRS) can be used to detect specific DNA sequences by methods based on hybridisation of oligonucleotide functionalised nanoparticles to a complementary DNA strand. In order to obtain a strong SERRS signal the analyte has to adsorb onto a suitable, roughened metal surface and a visible chromophore, which is resonant with the excitation frequency of the light and plasmon of the metal surface, has to be present in the molecule of interest. The problem which has to be overcome to use this technique for the detection of specific DNA sequences is that DNA is not SERRS active due to the lack of a visible chromophore and presence of the highly negatively charged phosphate backbone. To obtain SERRS active DNA a label containing a surface seeking group, to allow adsorption of DNA on to a metal surface, and a visible chromophore has to be attached to the DNA strand. This thesis reports the synthesis of four linkers containing a: fluorescent (aminofluorescein, fluorescein, TAMRA) or nou-fluorescent (BHQ) Raman tag, a surface complexing group (cyclic disulphide-thioctic acid) and a chemical functionality for the attachment of DNA (carboxyl group). Each of the linkers also contained poly(ethylene glycol) (PEG) (3 or 41 mer) which reduces the non-specific adsorption of molecules to nanoparticles surface and provides colloidal stability. The synthesised linkers were used to functionalize the following type's M metallic nanoparticles: Au citrate stabilised (18 and 50 nm), Ag citrate stabilised (40nm) and Ag EDTA stabilised (35nm). All prepared conjugates gave good SERRS responses at laser excitation frequencies of 633 nm and also exhibit high stability- they could be stored at room temperature for several weeks without any changes observed. In order to conjugate the prepared linker functionalized nanoparticles to oligonucleotides, the linker terminal COOH groups were reacted with amino-modified single stranded DNA in the presence of one of the coupling agents (N-(3- dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride-EDCHCl with N- hydroxysulfosuccinimide or 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4- methylmorpholinium chloride -DMT MM). This produced probes with a permanent SERRS signal which were then used for the detection of specific DNA sequences in a sandwich assay format. Surface enhanced Raman scattering (SERS) can also be a useful tool capable of providing information regarding changes to the chemical environment inside living cells, which would allow better understanding of the biochemistry of diseases. For example, it was reported that acidification of endosomes play a central role in a number of pathologies, including cystic fibrosis, kidney diseases and certain types of cancer. It has been reported that metallic nanoparticles, when incubated with the cells, are taken up by the cells, then trapped in endosomes. Formation of SERS active, pH sensitive metallic nanoparticle probes would allow the detection of changes of endosomal pH. In order to prepare such probes Au (citrate stabilised, 18 nm) and Ag (citrate and EDTA stabilised) nanoparticles were functionalised with two different pH sensitive molecules: 4-mercaptopyridine and 2-aminothiophenol. Ag EDTA nanoparticles functionalized with 4-mercaptopyridine gave very strong SERS signals and were found to be the most sensitive among all the pH sensing probe prepared. This probe was used to detect intracellular pH inside macrophages and HeLa cells. Because it was reported that the attachment of cell penetrating peptide or nuclear localization sequences (NLS) to nanoparticle surfaces makes it possible to deliver nanoparticles to other than endosomes components of a cell, a mixed monolayer of pH sensitive molecule (4-mercaptopyridine) and multifunctionallinker, able to attach to metal surface and to react with biomolecules of interest, was formed on gold and silver nanoparticles surface. It was found that pH sensitive probes prepared in such a way are the source of strong SERS signals and have the same sensitivity as metallic nanoparticles functionalised with 4-mercaptopyridine only. The prepared pH sensitive probe can be conjugated to NLS and used to detect pH inside the cell nucleus.

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La technique de spectroscopie basée sur la diffusion Raman Stokes spontanée est un procédé standard employé dans de nombreux domaines allant de la thermodynamique à la médecine, en passant par la science des matériaux. À la faveur d'un échange d'énergie inélastique, elle permet de déterminer les fréquences des vibrations moléculaires présentes dans un objet. On peut ainsi remonter à l'identification des molécules et ainsi caractériser l'objet d'étude sans utiliser de marqueur spécifique. Cette méthode est néanmoins affligée de défauts. Outre la présence d'un signal de fluorescence qui peut submerger la réponse Raman, le désavantage majeur est le long temps d'exposition que requière cette technique. Dans le cas d'étude d'échantillon biologique, cela proscris son usage pour des mesures de microspectroscopie : la cartographie spectrale d'objet microscopique. Afin de pallier ce problème, de nouvelles techniques ont été développées. C'est le cas de la spectroscopie employant la diffusion Raman anti-Stokes Cohérente (ou CARS pour Coherent Anti-Stokes Raman Scattering). Du fait de sa cohérence et de sa directivité le signal anti-Stokes affiche une intensité 10^5 to 10^6 fois plus importante que dans le cas de la diffusion Raman spontanée, ce qui permet alors d'abaisser le temps d'exposition à un niveau tolérable pour les objets biologiques lors d'une mesure de microspectroscopie. De plus, le caractère anti-Stokes du signal l'épargne de la contribution de la fluorescence. Pourtant, un défaut majeur limite encore l'utilisation de cette technique : le bruit de fond non résonant. Ce phénomène peut diminuer, voir noyer la contribution résonante qui porte l'information. Cette thèse a permis le développement de techniques CARS autorisant une réduction du bruit de fond non résonant. Pour ce faire un dispositif de spectroscopie CARS multiplex (M-CARS) en configuration copropagative a été construit. Ses capacités sont illustrées par des mesures spectrales d'échantillons minéral, végétal et biologique. À partir de ce système, il a été établi une méthode innovante permettant de discriminer le signal résonant du bruit non résonant en utilisant un champ électrique continu. Il est aussi démontré la mise en place d'un procédé qui a permis de mener la première mesure de microspectroscopie M-CARS en configuration contrapropagative sur un échantillon biologique. Cette configuration limite la collecte du signal à l'objet d'étude, empêchant ainsi l'acquisition du signal résonant et non résonant issu du solvant, principal responsable du bruit de fond non résonant lors d'une mesure CARS en configuration copropagative. / The spectroscopy technique based on spontanée Raman Stokes scattering is a standard process used in many fields spanning from thermodynamic and medicine, to materials sciences. An inelastic energy exchange permits to determinate the frequency of the molecular vibrations in an object. One can identify the molecules and thus, can characterize the object of study in a label-free way. Nevertheless, this method is afflicted with faults. Beside the presence of fluorecence that can drown the Raman answer, the main drawback is the long exposition time required. In the case of biological sample, this can prohibit the use of spontaneous Raman scattering for microspectroscopy measures: the spectral mapping of microscopic objects. To avoid this problem, new techniques have been developed. It is the case of Coherent anti-Stokes Raman scattering (CARS) spectroscopy. Due to its coherence and its directivity, the anti-Stokes signal has an intensity 105 to 106 times greater than the spontaneous Raman scattering one. The exposition time is then reduced to a tolerable level for biological objects during microspectroscopy measures. Moreover, the anti-Stokes characteristic of the signal prevents the fluorescence contribution. However, a major fault still limits the use of this technique: the nonresonant background. This phenomenon can diminish, even overwhelm the resonant contribution carrying the information. This thesis permitted the development of CARS approaches that allow the reduction of the nonresonant background. To do so, a multiplex CARS (M-CARS) spectroscopy apparatus in a forward configuration has been built. Its abilities are illustrated with spectral measures of mineral, vegetal and biological samples. Based on this system, it has been established an innovative method that can discriminate the resonant signal from the nonresonant one thanks to a static electric field. It has been also been demonstrated the development of a process that has allowed the first M-CARS microspectroscopy measure of a biological sample in a contrapropagative configuration. This setup limits the collect of the signal to the object of study, avoiding the acquisition of the resonant and resonant signals coming from the solvent, responsible for the major part of non resonant background during a CARS measure in a forward configuration.