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Détermination de la constante de dissociation de l'hybridation ADN / ADN aux interfaces solide/liquide par trois techniques différentes : résonance de plasmon de surface, biopuce par mesure de cartographie de fluorescence et par mesure de fluorescence par champ évanescent

Muchen Li 1
1 INL - C&N - INL - Chimie et Nanobiotechnologies
INL - Institut des Nanotechnologies de Lyon
Abstract : Biosensors are powerful detection and analysis tools that have been widely applied in pharmaceuticals, healthcare, biomedical research, and environmental monitoring. However different biosensors use different transducers and therefore different substrates and surface chemistries. All of these parameters may have an effect on the biomolecular reactions at the interface and lead to a deviation in dissociation constant Kd measurements. In this context, this PhD work aimed at comparing three different techniques: fluorescent microarray, evanescent field fluorescence biosensor and surface plasmon resonance (SPR) biosensor, to determine DNA hybridization Kd. For the classical fluorescence microarray, the substrate is a glass slide and the detection is performed at the end of the experiment. In the case of evanescent field fluorescence biosensor, polystyrene is the substrate and it permits a real-time detection. SPR is performed on thin gold film. It is a real-time and a label-free technique. The two fluorescent based techniques require to label the targets with fluorescent dyes prior to the measurements. One important factor determining the performance of the analysis is the surface chemistry of the sensor chip. Herein, we have optimized gold surface chemistry for thiol modified DNA grafting. We studied two cleaning methods: piranha solution and oxygen plasma, aiming at obtaining a clean gold surface without oxidation of the gold. Then, we optimized SPR assay parameters such as interfacial structure of sensor chip, ionic strength... After, these three techniques were used to measure the DNA hybridization Kd. The results showed that the Kds measured are similar for the three techniques. In addition, increasing surface probe density resulted in an increase of Kd of DNA hybridization.
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https://tel.archives-ouvertes.fr/tel-02052875
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Submitted on : Thursday, February 28, 2019 - 6:03:07 PM
Last modification on : Wednesday, July 8, 2020 - 12:44:07 PM
Long-term archiving on: : Wednesday, May 29, 2019 - 6:50:56 PM

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  • HAL Id : tel-02052875, version 1

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Muchen Li. Détermination de la constante de dissociation de l'hybridation ADN / ADN aux interfaces solide/liquide par trois techniques différentes : résonance de plasmon de surface, biopuce par mesure de cartographie de fluorescence et par mesure de fluorescence par champ évanescent. Autre. Université de Lyon, 2018. Français. ⟨NNT : 2018LYSEC028⟩. ⟨tel-02052875⟩

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