Lynn Mouawad

Développement de biocapteurs à cholinestérases pour la détection sélective d'ammoniums quaternaires: La mise en oeuvre de réglementations de plus en plus strictes en matière de sécurité alimentaire et environnementale a suscité l'intérêt pour les technologies des capteurs. Ces dispositifs analytiques permettent la détection rapide d'une large gamme de marqueurs (pathogènes, contaminants chimiques, etc.) comme alternatives aux techniques conventionnelles qui font face à diverses limitations. Les travaux présentés dans ce manuscrit ont porté sur le développement d'un biocapteur électrochimique basé sur l'inhibition des cholinestérases pour la détection des biocides, principalement le chlorure de benzalkonium (BAC) et le chlorure de didécyldiméthylammonium (DDAC). Dans un premier temps, un essai colorimétrique miniaturisé basé sur la réaction d'Ellman a été développé pour déterminer les effets inhibiteurs du BAC et du DDAC sur quatre cholinestérases issues de drosophile (DM AChE), d'anguille électrique (EE AChE), d'érythrocytes de boeuf (BE AChE), et de sérum de cheval (BChE). Ces composés ont inhibé les quatre enzymes de manière compétitive, à l'exception du BAC qui a inhibé la DM AChE de manière non compétitive. D'autre part, un docking moléculaire a été réalisé pour étudier les interactions structurales entre les cibles et les enzymes et pour corréler les résultats théoriques et expérimentaux. Dans un deuxième temps, diverses approches d'optimisation, telles que la détermination de la quantité optimale d'enzyme immobilisée à la surface de l'électrode, la température optimale de fonctionnement de l'enzyme et le type de cholinestérase le plus sensible, etc. ont été réalisées pour la détection sensible du BAC et du DDAC dans les eaux de lavage conformément aux normes imposées parl'Union européenne (UE). Ces résultats ont montré que les capteurs à base de BChE et de DM AChE ayant une concentration enzymatique de 0.9 mU par électrode étaient les plus sensibles au BAC et au DDAC, respectivement, à une température de 30°C. Cependant, les limites de détection enregistrées par les biocapteurs les plus sensibles de 0.32 µM pour le BAC et de 0.038 µM pour le DDAC n'étaient pas compatibles avec le seuil réglementaire pour les eaux de lavage, fixé à 0.028 µM. Par conséquent, une étape de préconcentration par extraction en phase solide a été nécessaire pour atteindre les normes imposées par l'UE. Les insecticides organophosphorés étant de puissants inhibiteurs des cholinestérases, leur présence éventuelle dans les eaux de lavage peut induire des réponses faussement positives des biocapteurs. Nous avons donc mis au point une méthode permettant de s'affranchir de ce problème en utilisant l'enzyme phosphotriestérase (PTE), qui hydrolyse efficacement les organophosphorés et améliore donc la sélectivité du biocapteur. Dans une troisième étape, les biocapteurs optimisés ont été utilisés pour détecter les cibles (BAC et DDAC) directement dans des échantillons de lait, conformément à lavaleur limite de 0.28 µM imposée par l'UE. Cependant, en raison de la présence d'effets de matrice causés par la phase lipidique et d'autres macromolécules présentes dans le lait, une extraction liquide-liquide permettant d'éliminer ces substances a dû être réalisée en amont de l'analyse par biocapteur.

A sensitive and reliable electrochemical biosensor for the detection of benzalkonium chloride (BAC) and didecyldimethylammonium chloride (DDAC), the most commonly used disinfectant biocides in the agri-food industry, is described. Acetylcholinesterase from Drosophila melanogaster (DM AChE) and butyrylcholinesterase from horse serum (BChE) were immobilized by entrapment in a photocrosslinkable polymer on the surface of carbon screen-printed electrodes. Preliminary tests conducted in phosphate buffer showed limits of detection (LODs) of 0.26 µM for BAC using the BChE-based sensor and 0.04 µM for DDAC using the DM AChE sensor. These performances comply with the European regulation for dairy products, which sets a maximum allowable concentration of 0.28 µM for biocides. However, when tested directly in milk samples, a dramatic decrease in the sensitivity of both sensors towards BAC and DDAC biocides was reported. To overcome this problem, a simple liquid–liquid extraction was necessary prior to biosensor measurements, ensuring that the biosensors met European regulatory standards and provided an unbiased response.

A sensitive and robust electrochemical cholinesterase-based sensor was developed to detect the quaternary ammonium (QAs) biocides most frequently found in agri-food industry wash waters: benzalkonium chloride (BAC) and didecyldimethylammonium chloride (DDAC). To reach the maximum residue limit of 28 nM imposed by the European Union (EU), two types of cholinesterases were tested, acetylcholinesterase (AChE, from Drosophila melanogaster) and butyrylcholinesterase (BChE, from horse serum). The sensors were designed by entrapping AChE or BChE on cobalt phthalocyanine-modified screen-printed carbon electrodes. The limits of detection (LOD) of the resulting biosensors were 38 nM for DDAC and 320 nM for BAC, using, respectively, AChE and BChE. A simple solid-phase extraction step was used to concentrate the samples before biosensor analysis, allowing for the accurate determination of DDAC and BAC in tap water with limits of quantification (LOQ) as low as 2.7 nM and 5.3 nM, respectively. Additional assays demonstrated that the use of a phosphotriesterase enzyme allows for the total removal of interferences due to the possible presence of organophosphate insecticides in the sample. The developed biosensors were shown to be stable during 3 months storage at 4 °C.

A sensitive and robust electrochemical cholinesterase-based sensor was developed to detect the quaternary ammonium (QAs) biocides most frequently found in agri-food industry wash waters: benzalkonium chloride (BAC) and didecyldimethylamonium chloride (DDAC). To reach the maximum residue limit of 2.8 10-8 M imposed by the European Union (EU), two types of cholinesterases were tested, acetylcholinesterase (AChE, from Drosophila melanogaster) and butyrylcholinesterase (BChE, from horse serum). The sensors were designed by entrapping AChE or BChE on cobalt-phthalocyanine-modified screen-printed carbon electrodes. The limits of detection (LOD) of the resulting biosensors were 3.8 10-8 M for DDAC and 3.2 10-7 M for BAC, using respectively AChE and BChE. A simple solid phase extraction step was used to concentrate the samples before biosensor analysis, allowing the accurate determination of DDAC and BAC in tap water with limits of quantification (LOQ) as low as 1.3 10-9 M and 5.3 10-9 M, respectively. The biosensor was shown to be stable during 3-months storage at 4°C.

Bipolar disorder is known to be a group of affective disorders depicted by depressive manic or hypomanic disorders. Indeed, considered as an oldie as its pharmaceutical usage started in the 19th -century lithium is also a goodie used as the first-line treatment for bipolar disorder, also it is considered to be the only treatment with anti-suicidal effects. During treatment, lithium levels should be monitored as its therapeutic levels (0.5–0.8 mM) in the blood are close to intoxication levels (>1.5 mM) and to severe intoxication levels >2.5 mM that might induce coma. That is why it is necessary for patients diagnosed with bipolar disorder to monitor lithium levels frequently. In fact, there are many traditional analytical techniques for lithium detection. Still, these are associated with limitations as they are very expensive, time-consuming, and not found in all laboratories. So, the need to develop a cost-effective, sensitive, and easy-to-handle devices has grown. Affinity sensors constituted a promising potential for the sensitive detection of lithium. This review, to my knowledge, is the first review highlighting the different types of biosensors developed for lithium-ion detection while explaining the mode of action of each biosensor, and talking about all possible biological fluids that can be used for the detection of this drug.

Breast cancer is the leading cancer type for women with two million new yearly infections and more than half a million dead worldwide. Human Epidermal Receptor 2 (HER2) is a prominent breast cancer biomarker that indicates aggressive cancer and is often associated with a bad prognosis and low survival rates. However, current detection methods for HER2 are often time-consuming, expensive, and require a high level of expertise. Biosensors are devices that turn biological interaction into a readable electronic signal; they are known for their high specificity and selectivity for low concentration, as well as their low cost and ease of use, thus making them a better alternative to traditional methods. Also, saliva is becoming a better alternative to blood for the detection of biomarkers due to its non-invasive collection in large quantities with simple collection methods with a richness in disease biomarkers including HER2. Thus, this project aims to develop a label-free, low cost, electrochemical biosensor for the detection of HER2 in saliva. This was done by first depositing diazonium salt onto a screen-printed electrode (SPE) through cyclic voltammetry, then immobilizing anti-HER2 antibodies on the activated SPE using 1-ethyl-3-(3-dimethylamino) propyl carbodiimide/N-hydroxysuccinimide. HER2 biomarker concentrations were detected using electrochemical impedance spectroscopy inside a microfluidic system. The biosensor showed a higher linear detection of HER2 (Y = 0.0062X + 0.1066/R² = 0.9909) in its physiological concentration range of 5 and 40 pg/mL when compared to other interference proteins: Epidermal Growth Factor Receptor (Y = 0.0016X + 0.0188/R² = 0.8072) and Human Epidermal Receptor 3 (Y = (0.0035X + 0.0225/R² = 0.1302). The biosensor was then used to detect 10 pg/mL of HER2 concentration in real saliva using the standard addition methods (Y = 0.0118X + 0.1282/R² = 0.9876).