Thèse soutenue

Immunocapteur à détection électrochimique directe et sans marqueur
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Auteur / Autrice : Hoang Vinh Tran
Direction : Minh Chau PhamBenoît Piro
Type : Thèse de doctorat
Discipline(s) : Surfaces, interfaces, matériaux fonctionnels
Date : Soutenance en 2013
Etablissement(s) : Paris 7

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In this work, we focused on design and fabrication of label-free and reagentless electrochemical biosensors based on multifunctional conducting polymers used as both electrochemical transducers and anchoring surfaces for the capture probes. Two applications of electrochemical biosensors have been developed: environment monitoring and nucleic acid detection. In the first application, we developed an original reagentless and label-free electrochemical biosensor for pesticide detection and made the proof-of-concept with the most widely used herbicide, atrazine. For this, we designed and synthesized a novel electropolymerizable 5-hydroxy¬1,4-naphthoquinone monomer coupled to hydroxyatrazine, [N-(6-(4-hydroxy-6-isopropylamino¬1,3,5 -triazin-2-ylamino)hexyl) 5 -hydroxy-1,4-naphthoquinone-3 -prop ionam ide] (JUG-HATZ). This monomer contains three functional groups: the hydroxyl group for electropolymerization, quinone for its electroactivity in aqueous medium and transduction capability, and the hydroxyatrazine moiety as capture probe for the anti-atrazine antibody (a-ATZ). Poly(JUG-HATZ) was obtained by electrooxidation of the corresponding monomer. Square wave voltammetry (SWV) shows that a glassy carbon electrode modified with this polymer presents a current decrease following a-ATZ complexation. By means of a competitive displacement, a highly specific current increase (signal-on) was obtained upon atrazine addition in solution. This constitutes a direct, label-free, reagentless and signal-on electrochemical immunosensor, with an excellent selectivity and a very low detection limit of ca. 1 pM, i. E. 0. 2 ng L-1, one of the lowest ever reported for an atrazine electrochemical sensor. This is far lower than the detection limit required by the European Union directives for drinkable water and food samples (0. 1 ug L-1) and the detection simplicity gives hope for a practical application in the industry. In the second application, we reported a novel and simple electrochemical method based on conducting polymer for reagentless and label-free detection of miRNA. This label-free microRNA sensor used an efficient bifunctional conjugated copolymer poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-2-carboxyethy1-1,4-naphthoquinone) acting both as immobilizing and transducing element. The carboxylic group is used as anchoring site for DNA-NH2 probe through peptide bond and the quinone function as an immobilized redox group for probing biomolecular interactions. We used, to establish the proof-of-concept, three miRNA: miR141, miR-103 and miR-29b-1 that are implied in prostate, bladder and lung cancers, respectively. The selectivity of this microRNA sensor is remarkable with very few cross hybridization due to the fine control of experimental which avoids fal se-positive signais. The sensitivity of this system is good with a limit of detection of 650 fM. In order to enhance the sensitivity of this sensor, we nanostructured the conducting polymer by carbon nanotubes. Applied to detection of microRNA-141, this sensor gave an extremely low limit of detection of 10 fM and was also able to detect microRNA in diluted serum. We also demonstrated that reduced graphene oxide can be an excellent substrate to nanostructure poly(JUG-co-JUGA) films. This way, we obtained a limit of detection even lower than with carbon nanotubes, of ca. 5 fM. This sensitivity corresponds to the expectations for real-samples analysis. We started to collaborate with physicians to quantify miRNAs in bones and teeth. At last, we used for the first time RNA. DNA antibodies to develop an electrochemical immunosensor for microRNA detection. The use of antibodies to recognize miRNA. DNA hybrids present a considerable advantage: antibodies can be used to verify the results of the hybridization step. Indeed, we can implement this three detection mode for a single experiment: signal-on, signal-off then signal-on again. This procedure is very useful to verify the results because it provides a triple check.