Skip to Main content Skip to Navigation
Theses

Détection et quantification d’acides nucléiques par l’intercalation de sondes électro-actives appliquée à l’intégration d’approches d’amplifications isothermes in vitro

Abstract : Nucleic acids detection and quantification via electro-active probes intercalation applied to the integration of in-vitro isothermal amplification reactions. Detection and quantification of pathogenic agents through their DNA has become increasingly important in applications such as molecular diagnostics and food safety control to environmental monitoring. Routinely, these analyses are performed using optical quantitative PCR method by coupling the in vitro DNA amplification reaction PCR with fluorescent detection. However, it requires the use of a thermocycler as well as the integration of optical instrumentation leading to a bulky and expensive apparatus, unable to process turbid or colored samples. In order to circumvent these limitations, an alternative electrochemical monitoring method for PCR, based on the use of electro-active probes preferentially binding to double stranded DNA, has been proposed and developed at the LEM. A pre-industrial demonstrator developed in partnership with the start-up Easy Life Science allows the parallelization of real time electrochemical PCR measurements in a custom-made 48 wells plate. This work deals with the thermic regulation problem by replacing PCR by two alternatives isothermal DNA amplification methods. First, the LAMP (Loop Mediated Isothermal Amplification), known to be highly efficient and specific, is combined to an electrochemical detection through the use of a panel of redox probes in order to obtain analytical performances competing with the standard optical techniques. Secondly an isothermal amplification method with a simpler mechanism than LAMP, based on the recycling of the products of an HCR (Hybridization Chain Reaction), is also proposed. Finally, a new miniaturized plate integrating, to date, one hundred low volume (≤ μL) electrochemical cells as well as the corresponding potentiostat are presented. The novelty of this plate relies on its very low cost fabrication process based on the simple assembly of screen-printed films. Although the results presented were at room temperature, in the long term, it is envisioned to monitor isothermal amplification reactions in a “digital” manner
Document type :
Theses
Complete list of metadatas

Cited literature [237 references]  Display  Hide  Download

https://tel.archives-ouvertes.fr/tel-01989699
Contributor : Abes Star :  Contact
Submitted on : Tuesday, January 22, 2019 - 3:05:19 PM
Last modification on : Wednesday, October 21, 2020 - 9:40:04 AM

Identifiers

  • HAL Id : tel-01989699, version 1

Citation

Alexandra Martin. Détection et quantification d’acides nucléiques par l’intercalation de sondes électro-actives appliquée à l’intégration d’approches d’amplifications isothermes in vitro. Autre. Université Sorbonne Paris Cité, 2016. Français. ⟨NNT : 2016USPCC233⟩. ⟨tel-01989699⟩

Share

Metrics

Record views

91

Files downloads

167