Dépôt numérique
RECHERCHER

Electrochemical biosensors for foodborne contaminants based on aptamers and graphene materials.

Eissa, Shimaa Hassan Hassan (2015). Electrochemical biosensors for foodborne contaminants based on aptamers and graphene materials. Thèse. Québec, Université du Québec, Institut national de la recherche scientifique, Doctorat en sciences de l'énergie et des matériaux, 203 p.

[img]
Prévisualisation
PDF
Télécharger (7MB) | Prévisualisation

Résumé

La salubrité des aliments est un objectif majeur de santé global, et le contrôle de qualité des aliments est important pour les autorités et les professionnels du domaine agroalimentaire. La présence de niveaux élevés des contaminants d'origine alimentaire tels que les allergènes et les toxines dans les aliments représentent un problème majeur de santé publique qui nécessite l'élaboration d'outils efficaces pour leur détection. Malgré la sensibilité relativement élevée de certaines méthodes de détection utilisées présentement, ces dernières sont très laborieuses, nécessitent beaucoup de temps, exigent un personnel hautement qualifié et elles sont aussi couteuses. Ces limitations encouragent la recherche d’autres alternatives pour les appliquer dans un régime de surveillance réglementaire afin de garantir un niveau élevé de protection des consommateurs. Par conséquent, les biocapteurs sont apparus récemment comme une solution intéressante, plus particulièrement, les biocapteurs électrochimiques sont devenus un choix attractif en raison de leur très faible coût, haute sensibilité, facilité d'utilisation et leur possibilité de miniaturisation. Toutefois, il y a deux principaux défis auxquels sont confrontés les biocapteurs électrochimiques disponibles pour la détection des contaminants alimentaires. Tout d'abord, les stratégies de détection qui sont généralement utilisées pour obtenir la sensibilité requise sont sophistiquées, et le temps d’analyse est long. En second lieu, on peut nommer une faible spécificité, leur coût élevé ainsi qu’ une stabilité limitée. Pour relever ces défis, ce travail décrit le développement de plateformes à base de graphène en tant que transducteur et des aptamères d’ADN comme récepteurs de reconnaissance et explore leurs applications pour la détection de certains contaminants d'origine alimentaire.

Abstract

Food safety is a global health goal and food quality control is essential for authorities and professional players in the food supply chain. The presence of unsafe levels of foodborne contaminants such as allergens and toxins in food represents a growing public health problem that necessitates the development of efficient tools for their detection. Despite the relatively high sensitivity of some of the currently used detection methods, they are highly laborious, time consuming, require highly trained personnel and are expensive. These limitations encourage the research for alternative tools to be applied in a regulatory monitoring regime in order to guarantee a high level of consumer protection. Therefore, biosensors have appeared recently as interesting alternatives that exhibited potential applications for food quality analysis. Particularly, electrochemical biosensors have become an attractive choice due to their very low cost, high sensitivity, ease of use and capability of miniaturization. However, two main challenges are facing the wide applicability of the available electrochemical biosensors for the detection of food contaminants today. First, the sophisticated detection strategies which are used to obtain the required sensitivity, usually include a time consuming, costly labelling process and multiple reagents and washing steps. Second, the poor specificity of the available recognition receptors, their high cost as well as their limited stability are major disadvantages. To address these challenges, this work describes the development of novel, simple, sensitive, specific and low cost biosensing platforms for the detection of some foodborne contaminants, particularly allergens and toxins. Advances in nanobiotechnology and its integration in biosensors result in the development of novel sensing platforms with highly improved performance. New biorecognition elements and nanomaterial-based transduction systems are among those nanobiotechnological concepts that are revolutionising the development of electrochemical biosensors. Here, we explore the use of DNA aptamers as recognition receptors as well as graphene platforms as transducers in electrochemical biosensors for food analysis. First, a functionalization method of graphene electrodes was demonstrated by electrochemical reduction of in situ generated aryl diazonium salts in aqueous acidic solution. Two diazonium salts were utilised in order to show the versatility of this approach: nitrophenyl and carboxyphenyl diazonium salts. The electrochemical modification protocol was optimized in order to generate monolayer of aryl groups on the graphene surface without complete passivation of the electrode. Unlike the reported methods for graphene functionalization, we demonstrated here the ability of the electrografting of aryl diazonium salt to attach an organic film to the graphene surface in a controlled manner by choosing the suitable grafting protocol. Next, the functionalized graphene electrodes were then used to develop label-free electrochemical immunosensors for the milk allergen β-lactoglobulin (β-LG) as well as the egg allergen ovalbumin (OVA) showing high sensitivity. Moreover, the electrografting approach was then applied on chemical vapour deposition (CVD) grown monolayer graphene in order to enable detailed investigation and characterization of the modified electrodes and subsequently applied for impedimetric biosensing of ovalbumin. This first attempt to use functionalized CVD graphene in biosensing represents a proof of concept that can be extended to other biosensing applications. The carboxyphenyl modified graphene electrodes (CP-GSPE) were also exploited to develop a direct competitive voltammetric immunosensor for the detection of the shellfish toxin okadaic acid (OA). A competitive assay between OA and fixed concentration of okadaic acid-ovalbumin conjugate (OA-OVA) for immobilized antibodies on the CP-GSPE was employed for the detection of OA. The developed immunosensor allowed the sensitive detection of OA in PBS buffer. The matrix effect studied with spiked shellfish tissue extracts showed a good percentage of recovery and the method was also validated with certified reference mussel samples. Second, I successfully selected, identified and characterized DNA aptamers that bind with high affinity and specificity to OA and brevetoxin-2 (BTX-2), marine biotoxins that accumulate in shellfish. The aptamers were selected using systematic evolution of ligands by exponential enrichment (SELEX) and exhibited dissociation constants in the nanomolar range. The binding of target toxins to aptamer pools/sequences was monitored using fluorescence and electrochemical impedance spectroscopy (EIS) techniques. The aptamers with the highest affinities were then used for the fabrication of label-free electrochemical biosensors for the detection of OA and BTX-2. The selected aptamers offer promising alternatives to the currently employed antibodies and can be exploited in different detection assays for such small molecule toxins. Third, by integrating the high affinity and specificity of DNA aptamers with the carbon nanomaterial graphene, highly sensitive and selective aptasensor for Microcystin- Leucine, Arginine (MC-LR) was successfully developed. A specific DNA aptamer against MC-LR that did not show cross reactivity with Microcystin- Leucine, Alanine (MC-LA) and Microcystin- Tyrosine, Arginine (YR) has been utilized as a model. A facile strategy was used for the aptasensor fabrication based on the noncovalent assembly of DNA aptamer on GSPE. This new approach has led to a rapid, low-cost, sensitive and specific detection method for MC-LR in buffer and spiked fish extract samples, offering several advantages over previously reported methods. First, the aptasensor was fabricated without labelling, minimizing cost and complexity, as well as preserving the affinity of the aptamer to MC-LR. Second, the use of graphene electrodes has achieved good sensitivity, particularly when compared with other MC-LR sensors with sophisticated fabrication protocols and detection schemes. Lastly, this graphene-based aptasensor is highly specific to MC-LR, with selectivity against other microcystin congeners hardly achievable by previous attempts. Moreover, we demonstrated in this work that the mechanism of the detection was based on the conformation change in part of the aptamer sequence without complete release from the graphene surface. We believe that this finding is important for exploiting aptamers in the detection of other small molecules using graphene platforms in the future. Finally, for a better understanding of the behaviour of different graphene samples that can be used for biosensing, a systematic study have been performed in order to investigate to which extent the size of graphene oxide (GO) sheets influence their structural properties as well as their biosensing performance. Graphene oxide sheets with different size ranges were separated. The sheets were then characterized via atomic force microscopy (AFM), scanning electron microscopy (SEM), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). The biosensing performance of these samples was compared using DNA aptamer against MC-LR toxin as well as an antibody against β-LG in label-free detection format. We observed different trends between the size of GO sheets and the sensitivity of MC-LR aptasensors and β-LG immunosensors fabricated either using covalent attachment or physical adsorption. Our results demonstrate that controlling the size of GO sheets may have profound impact on their use in specific biosensing applications. Thus, my work lays solid foundations for the development of new electrochemical biosensors for foodborne contaminants based on graphene materials as transducers and aptamers as recognition receptors.

Type de document: Thèse
Directeur de mémoire/thèse: Tavares, Ana C.
Co-directeurs de mémoire/thèse: Siaj, Mohamed
Mots-clés libres: plateformes à base de graphène; aptamères d’ADN; contaminants d'origine alimentaire; biocapteurs électrochimiques
Centre: Centre Énergie Matériaux Télécommunications
Date de dépôt: 06 avr. 2016 20:19
Dernière modification: 08 avr. 2016 19:04
URI: http://espace.inrs.ca/id/eprint/3363

Actions (Identification requise)

Modifier la notice Modifier la notice