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Development of lab on a chip platforms for bacteria detection based on loop-mediated isothermal amplification.

Safavieh, Mohammadali (2015). Development of lab on a chip platforms for bacteria detection based on loop-mediated isothermal amplification. Mémoire. Québec, Université du Québec, Institut national de la recherche scientifique, Maîtrise en sciences de l'énergie et des matériaux, 206 p.

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Abstract

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Infectious disease accounts for nearly 50 million illness incidences around the world. Applications of lab on chip devices in conducting biomedical research have been drawn substantial interests from scientists and researchers all over the world. These lab-on-achip systems create clinically useful technologies and have a number of competitive advantages over the conventional biomedical instruments due to the reduced reagents/samples consumption, decreased analysis times and operational costs. In addition, these systems facilitate the development of the portable devices and the possibility of automatically performing multiple assay processes. Early detection and notification of pathogenic bacteria, is therefore of considerable significance in disease control. DNA-based amplification technique, known as a precise diagnostic tool for detecting pathogens, has presented a distinctive advantages over the conventional microbiological culture-based methods for pathogen detection such as high sensitivity, specificity and rapidity, especially in the detection of bacteria in samples containing low concentrations. over the past decade, loop-mediated isothermal amplification (LAMP) has caught significant attention as it is provides rapid amplification with high sensitivity and specificity of the target gene. In this thesis, various lab on a chip based platforms have been developed combining DNA LAMP amplification for detection and quantification of pathogens. In the first part, a microfluidic device designed for amplification of malB gene of E.coli bacteria followed by electrochemical detection of the amplified product. Through amplification time optimization, microfluidic chip could detect and quantify 48 cfu/ml of bacteria in 35 min employing immobilisation-free electrochemical transduction. In the second part of thesis, we have invented a cassette device for high throughput detection of various bacteria types (gram negative & gram positive). The cassette consists of two aluminum reels and a plastic ribbon, which has an array of chambers. one reel act as the provider of ribbon whilst the other one act as collector. The collector reel is connected to a heater to provide adequate temperatures for the amplification. LAMP solutions with E.coli (as gram negative model for bacteria) was applied to each reservoir and a plastic tape cover the chambers and rolls into the collector reel. After 1 hour - amplification of E.coli was detected using a colorimetric method employing Hydronaphthol blue (HNB) with a 30 CFU/ml limit of detection. The color of HNB changed from purple to blue in the presence of bacterial amplified product. Through procedure modification we were able to detect as low as 200 CFU/ml of S.aureus as an example gram-positive bacteria using Calcein. In the third part of thesis, the cassette’s ribbon has been modified by using flexible screen-printed electrode attached at the bottom of each chamber. Using Osmium redox and immobilisation-free of electrochemical technique, we could monitor the amplification of DNA of E.coli and S.aureus bacteria in a real-time analysis and quantification. In the forth part of thesis, a novel biosensor platform has been developed to detect the viability of bacteria using T4 bacteriophage and label free impedance spectroscopy. We have shown that we could monitor viability of bacteria in the range of 103 -109 CFU/ml, while using LAMP amplification and linear sweep voltammetry technique we could detect 102 CFU/ml within 40 min response time. In the last part of the thesis, we summarize our findings, and provide contribution of these researches to the knowledge as well as health. In addition, future possible path to the research work will be discussed.

Type de document: Mémoire
Directeur de mémoire/thèse: Vetrone, Fiorenzo
Informations complémentaires: Résumé avec symboles
Mots-clés libres: bacteria detection; loop-mediated isothermal amplification (LAMP); infectious diseases; amplification isothermique en boucles; maladies infetieuses; détection des bactéries;
Centre: Centre Énergie Matériaux Télécommunications
Date de dépôt: 11 févr. 2016 18:50
Dernière modification: 11 févr. 2016 18:50
URI: http://espace.inrs.ca/id/eprint/3302

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