A simple potentiometric biosensor based on carboxylesterase for the analysis of aspartame

A potentiometric aspartame biosensor was fabricated by simply depositing the carboxylesterase (CES)-bonded poly(n-butyl acrylate-n-acryloxysuccimide) [CES-poly(nBA-NAS)] microspheres on a Ag/AgCl screen-printed pH selective electrode. The pH transducer was made from non-plasticized polyacrylate membrane containing a hydrogen ionophore and lipophilic salt. The immobilized CES enzyme catalyzed the enzymatic hydrolysis of aspartame to L-aspartic acid (L-Asp), L-phenylalanine and methanol. Potentiometric determination of aspartame concentration was performed by quantifying the hydrogen ion concentration produced from L-Asp. The potentiometric determination of aspartame exhibited good selectivity with near Nernstian response. The sensitivity of the biosensor was closed to the Nernstian value, i.e., 50-52 mV decade-1 with a dynamic linear response range from 10-5 to 10-2 M and detection limit approaching 10-6 M. The aspartame biosensor demonstrated good repeatability and reproducibility with relative standard deviation (RSD) of 1.9% and 1.6%, respectively (n=3). The potentiometric aspartame biosensor was demonstrated to be reliable for determining aspartame content in sweetener samples and was comparable to the conventional high-performance liquid chromatography (HPLC) method for aspartame analysis

Analytical Methods for Determination of Non-Nutritive Sweeteners in Foodstuffs

Sweeteners have been used in food for centuries to increase both taste and appearance. However, the consumption of sweeteners, mainly sugars, has an adverse effect on human health when consumed in excessive doses for a certain period, including alteration in gut microbiota, obesity, and diabetes. Therefore, the application of non-nutritive sweeteners in foodstuffs has risen dramatically in the last decade to substitute sugars. These sweeteners are commonly recognized as high-intensity sweeteners because, in a lower amount, they could achieve the same sweetness of sugar. Regulatory authorities and supervisory agencies around the globe have established the maximum amount of these high-intensity sweeteners used in food products. While the regulation is getting tighter on the market to ensure food safety, reliable analytical methods are required to assist the surveillance in monitoring the use of high-intensity sweeteners. Hence, it is also necessary to comprehend the most appropriate method for rapid and effective analyses applied for quality control in food industries, surveillance and monitoring on the market, etc. Apart from various analytical methods discussed here, extraction techniques, as an essential step of sample preparation, are also highlighted. The proper procedure, efficiency, and the use of solvents are discussed in this review to assist in selecting a suitable extraction method for a food matrix. Single- and multianalyte analyses of sweeteners are also described, employing various regular techniques, such as HPLC, and advanced techniques. Furthermore, to support on-site surveillance of sweeteners' usage in food products on the market, non-destructive analytical methods that provide practical, fast, and relatively low-cost analysis are widely implemented

Porphyrins as Colorimetric Indicators for Detection and Identification of Chemical and Biological Agents

The objective of this study was to design sensor surfaces for rapid, real-time, optical detection of chemical/biological warfare agents and/or environmental pollutants that yield a minimum of false readings. Porphyrins were used as colorimetric indicators for transduction in surfaces using biological recognition elements such as enzymes and as combination recognition element/transducer in other surfaces. Immobilization protocols and assaying procedures were developed for each of the sensor surfaces. As a reversible, competitive inhibitor of enzymes, porphyrins can be used for identification and quantification of the presence of a substrate or another competitive inhibitor of the enzyme. This technique has been useful for development of glass surfaces for the detection of cholinesterase inhibitors such as organophosphate compounds and nerve agent simulants at parts per trillion levels using acetylcholinesterase, butyrylcholinesterase, and organophosphorous hydrolase as recognition elements. Evanescent wave aDepartment of Physic

Estudio de biosensores electroquímicos basados en inmovilización enzimática

Els biosensors electroquímics són dispositius d'anàlisi que combinen l'especificitat de les reaccions bioquímiques amb la capacitat analítica de les tècniques electroquímiques. Gràcies a aquesta combinació, és possible determinar de forma ràpida, sensible i fiable diferents analits en mostres amb matrius complexes. Per tot això, l'ús de biosensors és una alternativa als mètodes clàssics d'anàlisi per a realitzar processos de control de qualitat en diferents sectors industrials. En la present Tesi, s'han desenvolupat biosensors amperomètrics enzimàtics basats en la immobilització d’oxidases sobre una interfase electroquímica de nanotubs d'òxid de titani (IV) altament ordenats (TiO2NTAs). Per a això, s'han estudiat diferents processos d'immobilització d'enzims basats en captura polimèrica i en immobilització covalent, i s'han avaluat els paràmetres analítics dels biosensors desenvolupats. En als processos de captura polimèrica, s'ha estudiat l'ús de kappa-carragenina, 2-hidroxietilmetacrilat (HEMA) i quitosà. Aquests hidrogels s'han utilitzat per a la immobilització de l'enzim glucosa oxidasa (GOx) i s'ha observat que tant el HEMA com el quitosà generen un microentorn favorable per a la conservació de l'activitat biològica de l'enzim. banda Per a la immobilització covalent, s'ha utilitzat pentafluorofenilmetacrilat (PFM) generació amb l’objectiu de generar enllaços entre les molècules d'enzim i la superfície del transductor. Per a això, s'ha modificat la superfície de la interfase electroquímica TiO2NTAs mitjançant dues tècniques de plasma: polimerització de PFM i sembrat del mateix polímer. S'ha observat que la superfície polimeritzada de PFM (ppPFM) presenta major hidrofobicitat que la superfície en la qual s'ha realitzat el sembrat de PFM (pgPFM) i que això té influència en la conformació que adopten les molècules d'enzim. Mentre que en la superfície ppPFM predominen conformacions amb baixa activitat, en la superfície pgPFM la major part de la població de les molècules de GOx adopten conformacions amb activitat catalítica. Per aquests motius, els biosensors amb sembrat per plasma de PFM presenten major sensibilitat enfront de la presència de glucosa que els biosensors basats en la polimerització de PFM. Finalment, s'han desenvolupat biosensors amperomètrics de glucosa i de glutamat amb matrius d'immobilització polimèriques i covalents: Ti/TiO2NTAs/GOx/Quitosà, Ti/TiO2NTAs/HEMA-co-EGDA/pgPFM/GOx/Quitosà i Ti/TiO2NTAs/GmOx/Quitosà. S'han realitzat determinacions sobre mostres alimentàries emprant aquests biosensors i els resultats s'han comparat amb els obtinguts amb tècniques de referència.Los biosensores electroquímicos son dispositivos de análisis que combinan la especificidad de las reacciones bioquímicas con la capacidad analítica de las técnicas electroquímicas. Gracias a esta combinación, es posible determinar de forma rápida, sensible y fiable distintos analitos en muestras con matrices complejas. Por ello, el uso de biosensores es una alternativa a los métodos clásicos de análisis para realizar procesos de control de calidad en distintos sectores industriales. En la presente Tesis, se han desarrollado biosensores amperométricos enzimáticos basados en la inmovilización de oxidasas sobre una interfase electroquímica de nanotubos de óxido de titanio (IV) altamente ordenados (TiO2NTAs). Para ello, se han estudiado diferentes procesos de inmovilización de enzimas basados en captura polimérica y en inmovilización covalente, y se han evaluado los parámetros analíticos de los biosensores desarrollados. En los procesos de captura polimérica, se ha estudiado el uso de kappa-carragenina, 2-hidroxietilmetacrilato (HEMA) y quitosano. Estos hidrogeles se han utilizado para la inmovilización del enzima glucosa oxidasa (GOx) y se ha observado que tanto HEMA como quitosano generan un microentorno favorable para la conservación de la actividad del enzima. Para la inmovilización covalente, se ha utilizado pentafluorofenilmetacrilato (PFM) con el objetivo de generar enlaces entre las moléculas de enzima y la superficie del transductor. Para ello, se ha modificado la superficie de la interfase electroquímica TiO2NTAs mediante dos técnicas de plasma: polimerización de PFM y sembrado del mismo polímero. Se ha observado que la superficie polimerizada de PFM (ppPFM) presenta mayor hidrofobicidad que la superficie en la que se ha realizado el sembrado de PFM (pgPFM) y que ello tiene influencia en la conformación que adoptan las moléculas de enzima. Mientras que en la superficie ppPFM predominan conformaciones con baja actividad, en la superficie pgPFM la mayor parte de la población de las moléculas de GOx adoptan conformaciones con actividad catalítica. Por estos motivos, los biosensores con sembrado por plasma de PFM presentan mayor sensibilidad frente a la presencia de glucosa que los biosensores basados en la polimerización de PFM. Finalmente, se han desarrollado biosensores amperométricos de glucosa y de glutamato con matrices de inmovilización poliméricas y covalentes: Ti/TiO2NTAs/GOx/Quitosano, Ti/TiO2NTAs/HEMA-co-EGDA/pgPFM/GOx/Quitosano y Ti/TiO2NTAs/GmOx/Quitosano. Se han realizado determinaciones sobre muestras alimentarias empleando estos biosensores y los resultados se han comparado con los obtenidos con técnicas de referencia.Electrochemical biosensors are analytical devices that combine the specificity of biochemical recognition processes with the analytical power of electrochemical techniques. Consequently, it is possible to perform rapid, sensitive and reliable determinations of different analytes present in complex samples. For this reason, the use of biosensors is an alternative to classical analytical methods to perform quality control processes in different industrial sectors. In this work, we have developed enzymatic amperometric biosensors based on the immobilization of oxidases on an electrochemical interface of highly ordered titanium dioxide nanotubes array (TiO2NTAs). Thus, processes of enzyme immobilization based on polymeric entrapment and covalent immobilization have been studied. The analytical parameters of these biosensors have been evaluated. For polymeric entrapment processes, kappa-carrageenan, 2-hydroxyethyl methacrylate (HEMA) and chitosan have been studied as immobilization matrices. These hydrogels have been used for the immobilization of the enzyme glucose oxidase (GOx) and it has been observed that both, HEMA and chitosan, generate a favorable microenvironment for the conservation of the activity of the enzyme. For covalent immobilization, pentafluorophenylmethacrylate (PFM) has been used in order to generate bonds between the enzyme molecules and the surface of the transducer. Thus, the electrochemical interface TiO2NTAs has been modified by two plasma techniques: polymerization of PFM and grafting of the same polymer. It has been observed that the polymerized surface of PFM (ppPFM) has a higher hydrophobicity than the surface in which the PFM has been grafted (pgPFM). Hydrophobicity has influence on the adopted enzyme molecules conformation. On the ppPFM surface, conformations with low activity predominate, and on the pgPFM surface most of the population of GOx molecules adopt conformations with catalytic activity. For these reasons, the biosensors with plasma grafted PFM show higher sensitivity in presence of glucose than the biosensors based on the PFM polymerization. Finally, amperometric glucose and glutamate biosensors with polymeric and covalent immobilization matrices have been developed: Ti/TiO2NTAs/GOx/Chitosan, Ti/TiO2NTAs/HEMA-co-EGDA/pgPFM/GOx/Chitosan and Ti/TiO2NTAs/GmOx/Chitosan. These biosensors have been used to determine the glucose and glutamate content in different food samples. The results have been compared with those obtained with reference techniques

Développement d’un nouveau biocapteur enzymatique ultrasensible pour la détection conductimétrique de l’ochratoxine A dans l’huile d’olive

A new ultrasensitive enzymatic biosensor for the direct conductometric detection of ochratoxin A (OTA) has been developed in this work. Thermolysin (TLN), a peptidase, was chosen as recognition element. The proposed biosensor is based on TLN immobilization into a polyvinyl alcohol (PVA)/polyethylenimine (PEI) matrix containing gold nanoparticles (AuNPs) and cross-linked at the surface of gold interdigitated microelectrodes using glutaraldehyde vapor. Under optimal conditions (35 min cross-linking time, working pH of 7 and temperature of 25◦C), the biosensor response was linear up to 60 nM OTA and the limit of detection was 1 nM. This value was 700 times lower than the detection limit obtained using the more classical method based on enzyme cross-linking in the presence of bovine serum albumin (BSA). PVA/PEI hydrogel creates a very favorable aqueous environment for the enzyme. In addition, interactions between protonated amino groups of PEI and negative charges of both citrated AuNPs and thermolysin improve their dispersion in the polymer blend, favoring enzyme stabilization and accessibility to the substrate (OTA). The developed OTA biosensor was very reproducible and stable over a 30 days period when stored at 4◦C in 20 mM phosphate buffer between two measurements. The method was further evaluated using commercial doped olive oil samples. No pretreatment of the sample was needed for testing and no matrix effect was observed. Recovery values were close to 100%, demonstrating the suitability of the proposed method for OTA screening in olive oilDans ce travail, nous nous sommes intéressés au développement d’un nouveau biocapteur enzymatique ultrasensible pour la détection conductimétrique d’une mycotoxine, l’ochratoxine A (OTA). Une peptidase, la thermolysine (TLN), a été choisie comme élément de reconnaissance. Le biocapteur proposé est basé sur l’immobilisation de la TLN dans une matrice d’alcool polyvinylique (PVA)/polyéthylènimine (PEI) contenant des nanoparticules d’or et réticulée à la surface de microélectrodes interdigitées à l’aide de vapeurs de glutaraldéhyde. Dans les conditions optimales (35 min de réticulation, mesure à pH 7 et 25°C), la réponse du biocapteur est linéaire jusqu’à 60 nM et la limite de détection est de 1 nM. Cette valeur est 700 fois plus basse que celle obtenue en utilisant une méthode d’immobilisation basée sur la co-réticulation de la TLN en présence d’albumine de sérum bovin (BSA). La matrice PVA/PEI crée un environnement aqueux favorable à l’enzyme. Par ailleurs, les interactions entre les groupements amines protonés du PEI et les charges négatives des nanoparticules citratées et de la TLN améliore leur dispersion dans la matrice et favorise la stabilisation de l’enzyme et son accessibilité au substrat (OTA). Le biocapteur conductimétrique développé est très reproductible et stable pendant 30 jours lorsqu’il est stocké à 4°C dans du tampon phosphate 20 mM pH7 entre 2 mesures. Le biocapteur a ensuite été évalué sur des échantillons d’huile d’olive commerciale dopée. Aucun prétraitement de l’échantillon n’a été nécessaire et des taux de recouvrement proches de 100% ont été obtenus, démontrant l’absence d’effet de matric

Biocatalytic Tools for Enzymatic Cascades

The field of biocatalysis is receiving increased attention over the years, owing to the incontrovertible necessity for sustainable processes. As essential part of biocatalysis, enzymes fulfil the demand for greener reactions thanks to their valuable properties, while providing simpler synthetic routes with higher selectivity than the traditional hazardous methods. Potentially, enzymes are remarkable catalysts but most of the times, they require special optimization before being effectively applied at industrial scale, which translates into significant investment of work and time. In this regard, the combination of enzymes as tool to assist a reaction or to carry out particular functions can facilitate the process. A series of biocatalytic tools have been created in this work to tackle specific problems. Afterwards, they were tested to evaluate their efficacy during the utilization

Investigation of Volatile Organic Compounds (VOCs) released as a result of spoilage in whole broccoli, carrots, onions and potatoes with HS-SPME and GC-MS

Vegetable spoilage renders a product undesirable due to changes in sensory characteristics. The aim of this study was to investigate the change in the fingerprint of VOC composition that occur as a result of spoilage in broccoli, carrots, onions and potatoes. SPME and GC-MS techniques were used to identify and determine the relative abundance of VOC associated with both fresh and spoilt vegetables. Although a number of similar compounds were detected in varying quantities in the headspace of fresh and spoilt samples, certain compounds which were detected in the headspace of spoilt vegetables were however absent in fresh samples. Analysis of the headspace of fresh vegetables indicated the presence of a variety of alkanes, alkenes and terpenes. Among VOCs identified in the spoilt samples were dimethyl disulphide and dimethyl sulphide in broccoli; Ethyl propanoate and Butyl acetate in carrots; 1-Propanethioland 2-Hexyl-5-methyl-3(2H)-furanone in onions; and 2, 3-Butanediol in potatoes. The overall results of this study indicate the presence of VOCs that can serve as potential biomarkers for early detection of quality deterioration and in turn enhance operational and quality control decisions in the vegetable industry

Synthesis, electrodynamics and biosensor applications of novel sulphonated polyaniline nanocomposites

Philosophiae Doctor - PhDThe overall aim of this thesis was to prepare nanostructured more processable heteronuclear sulphonated polyanyline nanocomposites with electroconductive properties suitable for applications in biosensors. The sulphonated self-assembled polyaniline and derivatised polyaniline nanocomposites (SPAHs) were prepared by chemical oxidative polymerisation or electrical decomposition. The SPAHs prepared include those of polyaniline (PANi), poly-o-methoxyaniline (POMA) and poly-2.5 dimethoxyaniline (PDMA). Two types of sulphonic acids of heteronuclear aromatic hydrocarbons were used in the production of sulphonated SPAH composites. These were anthracene sulphonic acid (ASA) and naphthalene sulphonic acids (NSA) wich played both doping and surfactant roles.South Afric