Nanobiocapteur SERS à haute sensibilité et sélectivité : application au diagnostic précoce des maladies

L'objectif de cette thèse était le développement d'un biocapteur basé sur l'exploitation des propriétés optiques des nanostructures métalliques et plus particulièrement sur la diffusion Raman exaltée de surface (DRES)pour détecter de très faibles quantités de molécules. L'optimisation de l'exaltation Raman passe par une meilleure compréhension des propriétés plasmoniques des nanostructures, de l'influence de la géométrie(taille, forme et arrangement) des nanoparticules et de la nature du métal. De plus, le substrat DRES est ensuite recouvert d'un biorécepteur spécifique de l'analyte ciblé. Le biorécepteur utilisé dans notre biocapteurest un aptamère, un simple brin d’ADN, qui présente une forte affinité avec la molécule à détecter par la formation d'une structure secondaire spécifique. L'optimisation du signal du biocapteur nécessite également une meilleure compréhension des interactions biorécepteur/biomolécule. Nous avons donc étudié ces interactions par DRES et déterminé l'influence des conditions expérimentales (solution tampon, ajout d'un espaceur, environnement liquide ou sec...) sur l'orientation de l'aptamère et sur son interaction. La détermination des caractéristiques spectrales de l'aptamère nous a permis d'identifier l'interaction et de fournir une vue approfondie des mécanismes d'interaction. Ces résultats ouvrent la voie à une nouvelle stratégie de détection pour les biocapteurs basés sur la DRES en utilisant des aptamères.The objective of this thesis was the development of a biosensor based on the exploitation of the optical properties of metallic nanostructures and more especially on the surface enhanced Raman scattering (SERS) to detect very small quantities of molecules. The optimization of the Raman enhancement requires a better understanding of the plasmonic properties of the nanostructures, of the influence of the geometry (size, shape and arrangement) of the nanoparticles and of the nature of the metal. Moreover, the SERS substrate is then grafted with a bioreceptor specific to the targeted analyte. The bioreceptor used in our biosensor is anaptamer, a single strand of DNA, which has a strong affinity with the molecule to be detected by the formation of a specific secondary structure. The optimization of the biosensor signal also requires a better understanding of the bioreceptor/biomolecule interactions. We therefore studied these interactions by SERS and determined the influence of experimental conditions (buffer solution, addition of a spacer, liquid or dry environment...) on the orientation of the aptamer and on its interaction. The determination of the spectral characteristics of the aptamer allowed us to identify the interaction and to provide a deep insight on the interaction mechanisms. These results pave the way to a new kind of SERS-based biosensor using aptamers

Gold Nanocylinders on Gold Film as a Multi-spectral SERS Substrate

The surface enhanced Raman scattering (SERS) efficiency of gold nanocylinders deposited on gold thin film is studied. Exploiting the specific plasmonic properties of such substrates, we determine the influence of the nanocylinder diameter and the film thickness on the SERS signal at three different excitation wavelengths (532, 638 and 785 nm). We demonstrate that the highest signal is reached for the highest diameter of 250 nm due to coupling between the nanocylinders and for the lowest thickness (20 nm) as the excited plasmon is created at the interface between the gold and glass substrate. Moreover, even if we show that the highest SERS efficiency is obtained for an excitation wavelength of 638 nm, a large SERS signal can be obtained at all excitation wavelengths and on a wide spectral range. We demonstrate that it can be related with the nature of the plasmon (propagative plasmon excited through the nanocylinder grating) and with its angular dependence (tuning of the plasmon position with the excitation angle). Such an effect allows the excitation of plasmon on nearly the whole visible range, and paves the way to multispectral SERS substrates

New insight into the aptamer conformation and aptamer/protein interaction by surface-enhanced Raman scattering and multivariate statistical analysis

International audienceWe study the interaction between one aptamer and its analyte (the MnSOD protein) by the combination of surface-enhanced Raman scattering and multivariate statistical analysis. We observe the aptamer structure and its evolution during the interaction under different experimental conditions (in air or in buffer). Through the spectral treatment by principal component analysis of a large set of SERS data, we were able to probe the aptamer conformations and orientations relative to the surface assuming that the in-plane nucleoside modes are selectively enhanced. We demonstrate that the aptamer orientation and thus its flexibility rely strongly on the presence of a spacer of 15 thymines and on the experimental conditions with the aptamer lying on the surface in air and standing in the buffer. We reveal for the first time that the interaction with MnSOD induces a large loss of flexibility and freezes the aptamer structure in a single conformation