Academic literature on the topic 'Enzymatic sensor'

Early-stage uric acid (UA) abnormality detection is crucial for a healthy human. With the evolution of nanoscience, metal oxide nanostructure-based sensors have become a potential candidate for health monitoring due to their low-cost, easy-to-handle, and portability. Herein, we demonstrate the synthesis of puffy balls-like cobalt oxide nanostructure using a hydrothermal method and utilize them to modify the working electrode for non-enzymatic electrochemical sensor fabrication. The non-enzymatic electrochemical sensor was utilized for UA determination using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The puffy balls-shaped cobalt oxide nanostructure-modified glassy carbon (GC) electrode exhibited excellent electro-catalytic activity during UA detection. Interestingly, when we compared the sensitivity of non-enzymatic electrochemical UA sensors, the DPV technique resulted in high sensitivity (2158 µA/mM.cm2) compared to the CV technique (sensitivity = 307 µA/mM.cm2). The developed non-enzymatic electrochemical UA sensor showed good selectivity, stability, reproducibility, and applicability in the human serum. Moreover, this study indicates that the puffy balls-shaped cobalt oxide nanostructure can be utilized as electrode material for designing (bio)sensors to detect a specific analyte.

The essential disadvantages of conventional glucose enzymatic biosensors such as high fabrication cost, poor stability of enzymes, pH value-dependent, and dedicated limitations, have been increasing the attraction of non-enzymatic glucose sensors research. Beneficially, patients with diabetes could use this type of sensor as a fourth-generation of glucose sensors with a very low cost and high performance. We demonstrate the most common acceptable transducer for a non-enzymatic glucose biosensor with a brief description of how it works. The review describes the utilization of graphene and its composites as new materials for high-performance non-enzymatic glucose biosensors. The electrochemical properties of graphene and the electrochemical characterization using the cyclic voltammetry (CV) technique of electrocatalysis electrodes towards glucose oxidation have been summarized. A recent synthesis method of the graphene-based electrodes for non-enzymatic glucose sensors have been introduced along with this study. Finally, the electrochemical properties such as linearity, sensitivity, and the limit of detection (LOD) for each sensor are introduced with a comparison with each other to figure out their strengths and weaknesses.

Transdermal alcohol biosensors have the ability to detect the alcohol that emanates from the bloodstream and diffuses through the skin. However, previous biosensors have suffered from long-term fouling of the sensor element and drift in the resulting sensor readings over time. Here, we report a wearable alcohol sensor platform that solves the problem of sensor fouling by enabling drift-free signals in vivo for up to 24 h and an interchangeable cartridge connection that enables consecutive days of measurement. We demonstrate how alcohol oxidase enzyme and Prussian Blue can be combined to prevent baseline drift above 25 nA, enabling sensitive detection of transdermal alcohol. Laboratory characterization of the enzymatic alcohol sensor demonstrates that the sensor is mass-transfer-limited by a diffusion-limiting membrane of lower permeability than human skin and a linear sensor range between 0 mM and 50 mM. Further, we show continuous transdermal alcohol data recorded with a human subject for two consecutive days. The non-invasive sensor presented here is an objective alternative to the self-reports used commonly to quantify alcohol consumption in research studies.

The detection of glucose is crucial in the management of diabetes and other medical conditions but also crucial in a wide range of industries such as food and beverages. The development of glucose sensors in the past century has allowed diabetic patients to effectively manage their disease and has saved lives. First-generation glucose sensors have considerable limitations in sensitivity and selectivity which has spurred the development of more advanced approaches for both the medical and industrial sectors. The wide range of application areas has resulted in a range of materials and fabrication techniques to produce novel glucose sensors that have higher sensitivity and selectivity, lower cost, and are simpler to use. A major focus has been on the development of enzymatic electrochemical sensors, typically using glucose oxidase. However, non-enzymatic approaches using direct electrochemistry of glucose on noble metals are now a viable approach in glucose biosensor design. This review discusses the mechanisms of electrochemical glucose sensing with a focus on the different generations of enzymatic-based sensors, their recent advances, and provides an overview of the next generation of non-enzymatic sensors. Advancements in manufacturing techniques and materials are key in propelling the field of glucose sensing, however, significant limitations remain which are highlighted in this review and requires addressing to obtain a more stable, sensitive, selective, cost efficient, and real-time glucose sensor.

In this 21th century, the demand for glucose sensors in monitoring diabetes reaches a year-on-year peak due to the unhealthy lifestyle of society. Therefore, it is the utmost important task for scientists and researchers to develop a highly efficient and effective glucose sensor. However, conventional enzymatic glucose sensors have showed some drawbacks and the underlying issues faced by enzymatic glucose sensors are outlined in this paper. With the tremendous advancement of science and technology, the field of diabetes monitoring has evolved from enzymatic to nonenzymatic glucose sensor that heavily emphasized on the usage of nanomaterial. This transformation is supported by various justifications such as a better stability of nonenzymatic sensors towards the surrounding, higher sensitivity and ease of fabrication. Numerous materials including graphene, noble metals, (transition) metal oxides and composites have been explored for its potential in the development and performance improvement of nonenzymatic glucose sensors. This paper reviewed nonenzymatic glucose sensors, their mechanism of glucose oxidation and various promising graphene-based nanocomposite systems as well as the challenges and future perspectives of glucose biosensors.

Non-enzymatic glucose sensors have been extensively exploited recently. But the nanostructured non-enzymatic sensors often suffer from the aggregation of the nanoscale particles and poor conductivity of the composed metal oxides. In this work, a highly conductive one-dimensional carbon nanofilm coated TiO2 nanotube arrays (TiO2@C NTAs) were fabricated as the substrate. Copper oxide nanoparticles (CuOx NPs) were then deposited on the substrate to prepare CuOx NPs/TiO2@C NTAs as the glucose sensor. Under optimal conditions, the CuOx NPs/TiO2@C NTAs sensor shows a linear dependence on glucose concentration from 0.001 to 2.467 mM, with a sensitivity of 1155.68 μA mM−1 cm−2. The detection limit is 0.17 μM (S/N = 3). The prepared sensor exhibits high reproducibility and selectivity towards glucose determination, with minimal response to the coexistent species such as mannose, fructose, and 4-acetaminophenol, etc. Monitoring glucose from human serum sample has also been conducted, suggesting good reliability of this sensor.

Creatinine is the final metabolic product of creatine in muscles and a widely accepted biomarker for chronic kidney disease. In this work, we present a non-enzymatic sensor based on an electrochemical pretreated screen-printed carbon electrode (PTSPCE) with electrodeposited Cu nanoparticles (CuNPs). To function in a PoC format, the prepared PTSPCE/CuNPs non-enzymatic sensors were used as disposable elements in a portable potentiostat. The pretreatment using mild anodic and cathodic potentials in PBS resulted in an increased electroactive surface area and improved conductivity, confirmed by cyclic voltammetry and electrochemical impedance. Moreover, the detection through the CuNPs–creatinine interaction showed an enhanced performance in the PTSPCE surface compared to the bare electrode. The optimized PTSPCE/CuNPs sensor showed a linear working range from 10 to 160 μM (R2 = 0.995), a sensitivity of 0.2582 μA·μM−1 and an LOD of 0.1 μM. The sensor analytical parameters covered the requirements of creatinine detection in biofluids such as blood and saliva, with a low interference of common biomarkers such as urea, glucose, and uric acid. When evaluated in Fusayama/Meyer artificial saliva, the PTSPCE/CuNPs showed an average recovery rate of 116%. According to the observed results, the non-enzymatic PTSPCE/CuNPs sensor can potentially operate as a creatinine early screening system in PoC format.

In this work, enzymatic doped/undoped poly-silicon nanowire sensors with different lengths were fabricated using a top-down technique to measure glucose concentration. The sensitivity and resolution of these sensors correlate well with the dopant property and length of nanowire. Experimental results indicate that the resolution is proportional to the nanowire length and dopant concentration. However, the sensitivity is inversely proportional to the nanowire length. The optimum resolution can be better than 0.02 mg/dL for a doped type sensor with length of 3.5 μm. Furthermore, the proposed sensor was demonstrated for 30 applications with similar current-time response and showed good repeatability.

Perovskite and graphene-based nanocomposites have attracted much attention and been proven as promising candidates for both gas (H2S and NH3) and electrochemical (H2O2, CH3OH and glucose) sensor applications. In this review, the development of portable sensor devices on the sensitivity, selectivity, cost effectiveness, and electrode stability of chemical and electrochemical applications is summarized. The authors are mainly focused on the common analytes in gas sensors such as hydrogen sulfide, ammonia, and electrochemical sensors including non-enzymatic glucose, hydrazine, dopamine, and hydrogen peroxide. Finally, the article also addressed the stability of composite performance and outlined recent strategies for future sensor perspectives.

Na constante busca de novas estratégias para melhorar a atividade catalítica, foi que a começos do século passado, a síntese de nanopartículas de formato controlado, tornou-se em um dos acontecimentos que revolucionaram a abordagem catalítica da Química, criando assim a linha da nanociência, onde com a síntese de nanopartículas de formato ao nível nano, é possível controlar as propriedades catalíticas dos materiais a nível macroscópico. O presente trabalho apresenta, a síntese de nanopartículas de óxido cuproso (NPs-Cu2O) com faces cristalográficas controladas. Foi possível sintetizar estruturas cúbicas, esféricas, e octaédricas, sendo os cubos e octaedros os que possuem faces cristalográficas de tipo (100) e (111), respectivamente. Entretanto, as esferas possuem uma mistura entre ambas das faces. As propriedades catalíticas das NPs-Cu2O foram testadas eletroquimicamente mediante uma reação modelo de detecção de glicose. As NPs-Cu2O, foram sintetizadas em médio básico com cloreto de cobre (CuC12) como percursor, posteriormente com concentrações diferentes de cloridrato de hidroxilamina (NH2OHoHCI) foram obtidas NPs-Cu2O com estrutura cúbica, octaédrica e esférica. Posteriormente, foram imobilizadas numa superfície de eletrodo de carbono vítreo, mediante a técnica de casting. A oxidação catalítica da glicose, permitiu observar que o desempenho da estrutura cúbica fossesuperior, com uma sensibilidade de 442 ± 7 µA mM-1 cm-2, enquanto as estruturas esféricas e octaédricas foram de 165 ± 3 µA mM-1 cm-2 e 38 ± 1 µA mM-1 cm-2, respectivamente. Seguido as NPs-Cu2O, foram testadas na presença de Ácido Ascórbico (AA) e Ácido Úrico (UA), foi observado que os cubos possuem uma seletividade única, comparada com as outras estruturas. Dito comportamento foi estudado com Analise computacional (DFT), onde foi possível de observar que a distribuição entre átomos de Cobre e Oxigênio, determina a seletividade do material. Numa segunda etapa, para entender a importância da conservação estrutural e integridade morfológica, foram testadas as NPs-Cu2O, aos diferentes dias após de ser sintetizadas, observando claramente uma relação entre estrutura e atividade catalítico. Foi observado que nas estruturas cúbicas o deterioro foi maior em comparação com as outras estruturas, isto acompanhado mediante DFT, foi determinado que estrutura cúbica apresenta uma maior interação com o oxigênio, provocando assim, que a rápida transformação de Cu(I) para Cu(II), como CuO. Por último as NPs-Cu2O, foram testadas por espectroscopia de fotoelétrons excitados por raios X (XPS), este analise ajudou a compreender que o desempenho catalítico, não estava relacionado com a formação de Cu (III). Estes resultados foram apoiados pelos resultados obtidos pela espectroscopia de infravermelho in situ (FTIR), já que nessa análise foi possível de observar como o estabilizante (SDS), foi determinante em cada estrutura.
In the constant search for new strategies by advance of catalytic activities, was that at the beginning of the last century the synthesis of nanoparticles in a controlled format, became one of the events that revolutionized the catalytic approach of Chemistry, thus creating a line of nanoscience, where with the synthesis of nanoparticles of format at the nano level, it is possible to control catalytic properties of materiais at the macroscopic level. Consequently, the present work the synthesis of cuprous oxide nanoparticles (Cu2O-NPs), with crystallography faces welldefined. It was possible synthesize cubic, spherical and octahedral structure, the cubes and octahedrons being those having crystallographic faces of type (100) and (111), respectively. Meanwhile, the spheres have a mixture between both faces. The catalytic properties of Cu2O-NPs were electrochemically tested by a model glucose detection reaction. The Cu2O-NPs were synthetized in basic solution with cooper chlorate (CuCl2) like precursor, after with different concentration of hydroxylamine hydrochloride (NH2OH· HCl) were obtain cubic, spheres and octahedral structure. Posteriorly, were immobilized in a glassy carbon surface, through the technique of casting. The catalyst oxidation of glucose allowed observe that the performance of cubic structure was superior, with a sensibility of 442 ± 7 µA mM-1 cm-2, while the spheres and octahedral structure were 165 ± 3 µA mM-1 cm-2 e 38 ± 1 µA mM-1 cm-2, respectively. Following the Cu2O-NPs, they were tested in the presence of Ascorbic Acid (AA) and Uric Acid (UA), it was observed that the cubes have a unique selectivity compared to the other Cu2O-NPs structure. This behavior was studies with com putational analysis (DFT), where it was possible to observed that the distribution between copper and oxygen atoms determines the selectivity of material. In a second step, to understand the importance of structure conservation and morphological integrity, Cu2O-NPs were tested at different days after being synthesized, noting clearly a relation between structure and catalytic activity. It was observed that cubic structure the deterioration was greater in comparation with the other structures, this being accompanied by DFT, it was determinate that cubic structure show a greater interaction with the oxygen, thus provoking that rapid transformation of Cu (I) to Cu(II), like CuO. Finally, the Cu2O-NPs were tested by x-ray excited photoelectron spectroscopy (XPS), this analysis helped to understand the catalytic activity was not related to Cu (III) formation. These results were supported by those obtained by in situ (FTIR), since in this analysis it was possible to observe how the stabilizer (SDS) was determinant in each structure.

This thesis presents the achievements and scientific work conducted using a previously designed and fabricated 64 x 64-pixel ion camera with the use of a 0.35 μm CMOS technology. We used an array of Ion Sensitive Field Effect Transistors (ISFETs) to monitor and measure chemical and biochemical reactions in real time. The area of our observation was a 4.2 x 4.3 mm silicon chip while the actual ISFET array covered an area of 715.8 x 715.8 μm consisting of 4096 ISFET pixels in total with a 1 μm separation space among them. The ion sensitive layer, the locus where all reactions took place was a silicon nitride layer, the final top layer of the austriamicrosystems 0.35 μm CMOS technology used. Our final measurements presented an average sensitivity of 30 mV/pH. With the addition of extra layers we were able to monitor a 65 mV voltage difference during our experiments with glucose and hexokinase, whereas a difference of 85 mV was detected for a similar glucose reaction mentioned in literature, and a 55 mV voltage difference while performing photosynthesis experiments with a biofilm made from cyanobacteria, whereas a voltage difference of 33.7 mV was detected as presented in literature for a similar cyanobacterial species using voltamemtric methods for detection. To monitor our experiments PXIe-6358 measurement cards were used and measurements were controlled by LabVIEW software. The chip was packaged and encapsulated using a PGA-100 chip carrier and a two-component commercial epoxy. Printed circuit board (PCB) has also been previously designed to provide interface between the chip and the measurement cards.

Le domaine de la bioélectronique, qui couple l'électronique et la biologie, présente un fort potentiel pour le développement de nouveaux outils biomédicaux. Les dispositifs à base d’électronique organique sont particulièrement prometteurs; l'utilisation de ces matériaux organiques confère une interface idéale entre les mondes biologique et électronique en raison de leur biocompatibilité et de leur possible grande flexibilité. Le transistor électrochimique organique (OECT) représente un dispositif prometteur dans ce domaine. Des OECT ont par exemple été intégrés dans des systèmes permettant de détecter localement l’activité ionique/biomoléculaire, de mesurer l'activité d'une cellule unique, mais aussi d’effectuer la caractérisation de tissus et le suivi du fonctionnement d’organes entiers. L'OECT est un dispositif extrêmement polyvalent qui apparaît comme un outil thérapeutique et de diagnostic de première importance. L'utilisation de matériaux organiques tels que les polymères conducteurs, rend l‘OECT adaptable pour une large gamme d'applications. Un exemple représentatif est le capteur de glucose. L'OECT, en raison de ses propriétés d'amplification, peut augmenter ces courants de plusieurs ordres de grandeurs. Utilisé comme capteur de glucose, il montre une forte sensibilité et des limites de détection des concentrations de l’ordre du nanomolar. Cependant, en dehors d’une meilleure précision de mesure, la stabilité est nécessaire pour les applications à long terme. Par exemple, ces capteurs se doivent d'enregistrer en continu les variations de glycémie chez des personnes pendant plusieurs jours et sans défaillance. Le glucose est la source d'énergie principale du cerveau. Ainsi, l'enregistrement de la modulation des niveaux de glucose avant et/ou pendant la crise d'épilepsie peut donner beaucoup d'informations dans la compréhension de cette maladie. Pour des applications à long termes, une liaison covalente de la biomolécule est préférable.La biofonctionnalisation des polymères conducteurs, qui sont utilisés comme matières actives dans les OECTs, est une étape obligatoire qui mettra en évidence les propriétés de l’OECT telles que la biocompatibilité, la stabilité, et la fonctionnalité. Dans ce travail, des méthodes de biofonctionnalisation du poly (3,4-éthylènedioxythiophène) dopé avec des anions de tosylate (PEDOT: TOS) ou dopé avec du poly (styrène sulfonate) (PEDOT: PSS) ont été développéeset ont conduitsent à des améliorations telles que la biocompatibilité accrue avec les cellules et à une stabilité accrue pour les applications de détection. En outre, nous avons étudié l'utilisation de liquides ioniques en combinaison avec des polymères réticulables comme alternatives aux électrolytes conventionnelles. Ces gels ioniques électrolytes ont amélioré la stabilité des enregistrements électrophysiologiques. Enfin, des mesures in vitro de l'activité métabolique de la cellule ont été effectuées. Le suivi de l'absorption du glucose et de la conversion en lactate fournit des informations sur la santé des cellules et comment ses activités métaboliques sont affectées par la présence de composés toxiques et d’agents pathogènes
The rising field of bioelectronics, which couples the realms of electronics and biology, holds huge potential for the development of novel biomedical devices for therapeutics and diagnostics. Organic electronic devices are particularly promising; the use of robust organic electronic materials provides an ideal bio-interface due to their reported biocompatibility, and mechanical matching between the sensor element and the biological environment, are amongst the advantages unique to this class of materials. One promising device emerging from this field is the organic electrochemical transistor (OECT). The OECT combines properties and characteristics that can be tuned for a wide spectrum of biological applications. These applications have allowed the development of OECTs to sense local ionic/biomolecular and single cell activity, as well as characterization of tissue and even monitoring of function of whole organs. The OECT is an extremely versatile device that emerges as an important player for therapeutics and diagnostics.The use of organic materials, such as conducting polymers, makes the OECT tunable for a wide range of applications. For example, OECTs have been used for sensing applications. A representative example is the glucose sensor. The OECT has been used as glucose sensor and has shown high sensitivities and low limit of detection for concentrations at the nanomolar range. However, apart from high sensitivities, stability and reproducibility are common necessities for long term applications. For example, it is of equal importance for these sensors to continuously record variations of glucose for diabetic patients, since multiple measurements per day without failure are necessary. Additionally, stability is necessary for implantable sensors. For brain cells such as neurons, glucose is the main energy source. Thus recording modulations of glucose levels before or during an epileptic crisis will enhance our understanding of this disease. Long-term stabilities for these sensors can be achieved through biofunctionalization, which is a method to attach a biomolecule to a device. For long term applications a covalent binding of the biomolecule is preferred. Biofunctionalization of conducting polymers, which are used as active materials in OECTs, is a mandatory step that can enhance OECT properties such as biocompatibility, stability, and functionality. In this work, different biofunctionalization methods of poly(3,4-ethylenedioxythiophene) doped with tosylate anions (PEDOT:TOS) or doped with poly(styrene sulfonate) (PEDOT:PSS) have been explored. The biofunctionalization methods have led to improvements for different applications such as better interfaces with living cells, and better stability for enzymatic sensors. Additionally, we have employed the use of ionic liquids in combination with cross-linkable polymers as alternative solid state electrolytes. These electrolytes are improving the stability of recordings in electrophysiology. Finally, in vitro measurements of metabolic activities in cells have been explored. The monitoring of glucose uptake and its conversion to lactate is a sensitive indicator of the viability of these cells. Furthermore, in the presence of toxic compounds and pathogens, the nature or kinetics of these metabolic activities is getting affected. Therefore, OECTs used for glucose and lactate sensing can at the same time be used for Immunosensing

Molecular magnetic resonance imaging (MRI) methods have the potential to provide detailed information regarding cellular and molecular processes at small scales within the human body. Nuclear signals from chemical samples can be probed using specialised MRI techniques, to highlight molecular contrast from particular enzymes or metabolites. The aim of the work described in this thesis is to investigate both exogenous and endogenous contrast mechanisms using fluorine MRI and chemical exchange saturation transfer (CEST) respectively, in order to detect molecular changes in vitro. Initial theoretical work investigates the factors which affect fluorine MRI signals and provides a theoretical framework to determine the sensitivity of such experiments. A novel paramagnetic fluorine sensor to detect enzyme activity is then characterised using high field nuclear magnetic resonance (NMR), showing 60 to 70–fold increases in T1 relaxation values upon enzyme interaction. The effects on the fluorine lineshape from varying sample temperature and solvent were investigated. The possibility of imaging is demonstrated, but further investigations using the theoretical framework found pre–clinical implementation of the sensor is limited by the achievable experimental sensitivity. Efforts then focussed on CEST molecular methods, which are not limited by sensitivity. A protocol is developed to target amide protons in an in vitro cancer cell model, with parameters optimised following simulation of the expected contrast. Analysis of CEST results were aided through use of a support vector machine (SVM) to distinguish group differences between cancer cells and control samples. A linear classifier was found to be suitable to discriminate between samples.

Depuis 4 décennies, un modèle intermédiaire entre les traditionnelles approches in vivo et in vitro émerge : les Systèmes MicroPhysiologiques (SMP). Ils sont construits pour recréer différents niveaux de physiologie humaine, du simple organe à leurs interactions. Ils améliorent l’environnement de culture grâce à des microstructures accueillant des modèles d’architecture 3D et multicellulaire, et intègrent des microcapteurs monitorant l’activité cellulaire et leur environnement.Ce nouvel outil d’investigation est d’intérêt pour la recherche fondamentale sur les maladies comme le diabète. Dans le cas de cette maladie incurable, la régulation du glucose sanguin, résultant d’interactions complexes entre les îlots pancréatiques, le foie, les adipocytes et les muscles, est altérée. Un Multi-Organe-sur-Puce (MOsP) est un SMP pouvant reproduire ces interactions, et représente donc un modèle pertinent pour la recherche sur le diabète. En effet, la régulation inter-organe n’est pas entièrement reproduite par les modèles in vitro usuels, et requiert de multiples capteurs, ce qui est éthiquement et techniquement impossible in vivo. Dans le contexte du diabète, il n’existe aucun MOsPs reproduisant l’action des îlots sur les muscles, malgré l’importance des muscles squelettiques dans la régulation glycémique.Cette thèse propose une méthodologie pour construire un MOsP étudiant les interactions d’îlot à muscle dans la régulation glycémique. Les 3 objectifs du MOsP étaient : atteindre des concentrations physiologiques d’insuline grâce à des îlots sécrétant en réponse à une élévation physiologique de glucose, induisant une prise de glucose mesurable par les muscles, et monitorer l’expérience en direct. Pour cela, les investigations ont été menées avec une approche interdisciplinaire, utilisant et confrontant des résultats venant d’expériences biologiques in vitro et de simulations modélisant la biologie et la physique.Ce manuscrit détaille les étapes de la méthodologie, et délivre différents designs pour progressivement construire un MOsP comprenant: une puce microfluidique contenant les cellules et un capteur de glucose connecté directement au flux. Les principales découvertes ont été :- Un milieu et procédure de co-culture entre îlots primaires et LHCN-M2 myotubes ont été démontrés.- Un substrat de culture commun de type MicroElectrodes Array a été trouvé.- Des îlots ont été cultivés en puce microfluidique, et ont présenté une sécrétion d’insuline en réponse au glucose durant des expériences en fluidique. Des myotubes ont pu se différentier en puce, et ont présenté une prise de glucose basale (insuline indépendant).- Une stratégie in vitro-in silico pour dimensionner le MOsP a été développée et implémentée. Un modèle in silico simplifié d’îlot a été développé pour rapidement explorer 2 designs de puce. Des expériences in vitro correspondantes, de sécrétion d’insuline, ont été menées et confrontées aux expériences in silico. Les résultats ont soulevé l’hypothèse que les îlots n’avaient pas une fonctionnalité optimale dans nos petits volumes de culture. La même constatation a été faite concernant les myotubes, où la prise de glucose insuline dépendante a été démontrée en macro volumes, mais en micro volumes, la réponse observée (uniquement à concentration physiologique d’insuline) doit être reproduite avec des expériences plus robustes pour démontrer leur présence.- Un capteur de glucose compatible avec le système microfluidique a été caractérisé à l’aide d’expériences in vitro et in silico.- Un multi-potentiostat a été développé dans la perspective de futures mesures électrochimiques multiples et intégrées.Les bases et perspectives présentées ici permettront d’achever le MOsP îlot-muscle par de futurs travaux. La méthodologie peut aussi être réutilisée pour l’ajout de nouveaux organes (foie, adipocytes) complétant le MOsP, qui permettra de mieux comprendre les dérégulations intervenant dans le diabète de type 2
Over the past 4 decades, an intermediate model between the traditional in vivo and in vitro approaches has emerged: the MicroPhysiological Systems (MPS). MPS are designed to recapitulate different levels of human physiology, from the single organ to organs crosstalk. They upgrade the culture environment by patterning microstructures hosting 3D and multicellular architecture models and integrate microsensors monitoring cell activity and environment.This new investigation tool is of interest in fundamental research on diseases such as diabetes. In this incurable disease, blood glucose regulation, resulting from a complex organs interplay between the pancreatic islets, the liver, the adipocytes and the muscles, is impaired. A Multi-Organ-on-a-Chip (MOoC) is a MPS that can recapitulate these organs crosstalk and represents a relevant model for diabetes research. Indeed, inter-organ regulations are not recapitulated by usual in vitro models, and deciphering these interactions requires multiple sensors, which is not ethically and technically possible in vivo. In the context of diabetes, MOoCs reproducing the islets to skeletal muscles communication do not exist so far, despite the importance of the skeletal muscles impact on blood glucose, under islets action.In this thesis, we propose a methodology to design a MOoC deciphering islets to muscles interactions in blood glucose regulation. The MOoC objectives were to: (i) attain physiological insulin concentration secreted by islets in response to physiological glucose elevation, (ii) that induces a measurable glucose uptake by the muscle cells, (iii) monitor online relevant parameters. To that end, the investigations were conducted with an interdisciplinary approach, using and confronting results from both in vitro biological experiments and in silico modelling of biology and physics.This manuscript details the methodology steps, delivering different designs for progressive validation toward a complete MOoC that comprises a microfluidic chip with cells and an online glucose sensor. During the MOoC construction, our main findings were the following:- A co-culture medium and procedure for primary islets and LHCN-M2 myotubes were demonstrated.- A common MicroElectrodes Array-based substrate was found suited for co-culture in a single microfluidic chip.- Islets were cultured in microfluidic chips, and presented an insulin secretory response to glucose during fluidic experiments. Myotubes were successfully differentiated in microfluidic chips, and presented a measurable basal (insulin-independent) glucose uptake.- An in silico and in vitro informed MOoC scaling strategy was developed and implemented. A simplified in silico islet model was developed to rapidly explore chip designs. Corresponding in vitro insulin secretion experiments were conducted and confronted to the in silico experiments. Results raised the hypothesis that islets function was sub optimal when cultured in our low volume. Similar observation was made concerning myotubes scaling, where insulin-dependent glucose uptake was demonstrated in macro volumes experiments, but in micro volumes, the observed insulin response (only at physiological insulin concentration) has to be further repeated with improved experiments to explicitly demonstrate its presence.- A glucose biosensor compatible with microfluidic was characterized under different injection protocols, using in vitro and in silico experiments.- A multi-potentiostat was developed in the perspective of multiple and integrated electrochemical sensing in the MOoC.From the grounds and perspectives presented in this thesis, future work can be conducted to further complete this islet-muscle MOoC. The methodology can be re-used and extended in the perspective of adding new organs (liver, adipocytes) in this MOoC in order to better address the interorgan crosstalk deregulations in type 2 diabetes pathophysiology

Les tests sur le lieu de soins sont très prometteurs car ils permettent d'effectuer des mesures en temps réel et en continu à un prix abordable et s'adressent à un large éventail de personnes. Cependant, le défi que représente la surveillance continue pour gérer la santé de manière proactive tout en réduisant les dépenses de santé est considérable. Il s'agit principalement de garantir la fiabilité des éléments de reconnaissance et la viabilité à long terme des sources d'énergie, en particulier des batteries. Cette étude a établi un capteur de lactate non enzymatique pour les tests sur le lieu de soins, en utilisant une approche holistique qui comprend la modification de la morphologie de l'électrode, l'électrodéposition de matériaux conducteurs et de catalyseurs à l'échelle nanométrique, l'intégration d'un liquide ionique pour la sélectivité, l'optimisation de la technologie d'alimentation sans fil et l'incorporation de systèmes de gestion de l'énergie dans des dispositifs de détection électrochimique conçus par l'utilisateur lui-même. Les principaux résultats comprennent la sélectivité des catalyseurs non enzymatiques pour la détection et la proposition d'un dispositif de mesure personnalisé alimenté sans fil. Plus précisément, la modification de la géométrie du noyau du transformateur en ferrite a amélioré la puissance de sortie maximale du transducteur magnétoélectrique, qui a atteint 1,63 mW. Le circuit de gestion de l'énergie proposé fournit du courant continu avec un rendement élevé (0,74 mW) et permet une charge plus rapide pour la transmission d'énergie sans fil afin de soutenir nos dispositifs électrochimiques. Les dispositifs d'analyse électrochimique tels qu'ils ont été fabriqués ont démontré des capacités de mesure précises. L'utilisation de l'électrode poreuse imprimée a permis d'améliorer la reproductivité, la conductivité et la surface. L'électrodéposition de graphène et de nanoparticules de Ni(OH)₂, dont la taille et l'état chimique ont été soigneusement réglés, a augmenté la sensibilité du capteur. La plage de détection étendue des capteurs d'acide lactique optimisés s'avère avantageuse pour la détection du lactate, ce qui présente des avantages significatifs pour le diagnostic de diverses maladies. Un liquide ionique synthétisé sur mesure a facilité la détection sélective de l'acide lactique, en bloquant les molécules d'interférence et en permettant une détection "en une étape" avec une large gamme (1 mM à 60 mM) et une sensibilité élevée (1,374 μA/mM). En outre, la performance électrochimique du capteur non enzymatique avec liquide ionique a été étudiée en corrélant le coefficient de diffusion avec la Stokes-Einstein relationship. En conclusion, cette recherche offre des perspectives précieuses sur des systèmes de tests de soins au point d'intervention entièrement intégrés avec des applications pratiques, notamment les capteurs de lactate non enzymatiques avec des liquides ioniques et les transducteurs magnétoélectriques pour le transfert d'énergie sans fil. L'effort continu visant à améliorer les dispositifs de tests de soins au point d'intervention souligne l'importance de la recherche et de l'innovation soutenues pour faire progresser les soins aux patients et la gestion des maladies dans divers domaines, y compris la médecine clinique, la gestion du sport et la recherche sur le cancer
Point-of-care testing (POCT) holds great promise for providing real-time and continuous measurements at an affordable price, catering to a broad range of individuals. However, the challenge of continuous monitoring to proactively manage health while reducing healthcare expenses is substantial. These challenges primarily revolve around ensuring the reliability of recognition elements and the long-term sustainability of power sources, particularly batteries. This study established a non-enzymatic lactate sensor for point-of-care testing, employing a holistic approach that encompasses the modification of electrode morphology, electrodeposition of nanoscale conductive materials and catalysts, integration of ionic liquid for selectivity, optimization of wireless power supply technology, and the incorporation of power management systems into self-designed electrochemical detection devices. Key findings include conferring selectivity on non-enzymatic catalysts for detection and proposing a custom wirelessly supplied measurement device. Specifically, modifying the ferrite transformer core geometry improved the magnetoelectric transducer's maximum output power, reaching 1.63 mW. The proposed power management circuit supplied DC with high efficiency (0.74 mW) and enabled faster charging for wireless power transmission to support our electrochemical devices. The as-fabricated electrochemical analysis devices demonstrated precise measurement capabilities.Using the porous screening printed electrode showed increased reproductivity, conductivity, and surface area. The electrodeposition of graphene and Ni(OH)₂ nanoparticles, carefully regulated in size and chemical state, elevated the sensor's sensitivity. The extensive detection range of the optimized lactic acid sensors proves advantageous for detecting lactate, offering significant benefits in various disease diagnoses. A custom-synthesized ionic liquid facilitated selective detection of lactic acid, blocking interference molecules and enabling "1-step" detection with a wide range (1 mM to 60 mM) and high sensitivity (1.374 μA/mM). Additionally, the electrochemical performance of the non-enzymatic sensor with ionic liquid was investigated by correlating the diffusion coefficient with the Stokes-Einstein relationship. In conclusion, this research provides valuable insights into fully integrated POCT systems with practical applications, including the non-enzymatic lactate sensors with ionic liquids and magnetoelectric transducers for wireless power transfer. The ongoing effort to enhance POCT devices underscores the importance of sustained research and innovation in advancing patient care and disease management across various fields, including clinical medicine, sports management, and cancer research

[EN] Persimmon (Diospyros kaki L.) 'Rojo Brillante' is an astringent variety characterised by good growing conditions, excellent colour, size, sensory characteristics and good nutritional properties. In the last decade, its production has grown substantially in Spain given the application of high levels of CO2 to remove astringency while firmness is preserved. This technology has also increased its potential as a fresh-cut commodity. However, physical damage during processing result in degradation of the colour and firmness of the product and a higher susceptibility to microbial spoilage that significantly reduces the fruit's shelf life. The objective of the present thesis was to develop optimum procedures for processing and marketing 'Rojo Brillante' persimmon into a fresh-cut product with the maximum shelf life and best physicochemical, nutritional, sensory and microbiological quality. Firstly, the objective was to evaluate the effect of the maturity stage (MS) at harvest, storage time at 15 ºC before processing, and the application of different antioxidant treatments on enzymatic browning, sensory and nutritional quality of fresh-cut 'Rojo Brillante' persimmon during storage at 5 ºC. Concentrations of 10 g L-1 ascorbic acid (AA) or 10 g L-1 citric acid (CA) controlled tissue browning and maintained the visual quality of fresh-cut persimmon above the limit of marketability for 6-8 storage days at 5 ºC, depending on the MS. However, these acidic solutions reduced fruit firmness as compared to control samples. Further studies showed that the combination of these antioxidants with 10 g L-1 CaCl2 maintained firmness of the persimmon slices within the same range as the control samples. In another work, the application of 1-methylcyclopropene (1-MCP) allowed to process fruits after 45 days of storage at 1 ºC with commercial firmness and the antioxidant solution (10 g L-1 CA + 10 g L-1 CaCl2) extended the limit of marketability up to 9 days of storage at 5 ºC. Different controlled atmosphere conditions in combination with AA or CA dips were also evaluated as a first step to select optimum O2 and CO2 concentrations for modified atmosphere packaging (MAP) of fresh-cut 'Rojo Brillante' persimmons. Overall, the combination of antioxidant dips and a controlled atmosphere composed of 5 kPa O2 (balance N2) was proved to be the most effective combination to control enzymatic browning. This atmosphere maintained the visual quality of persimmon slices within the limit of marketability during 7- 9 days at 5 ºC. On the contrary, high CO2 concentrations (10 or 20 kPa) induced darkening in some tissue areas, associated with a flesh disorder known as 'internal flesh browning'. Later studies confirmed the beneficial effect of an active MAP in 5 kPa O2 compared to passive MAP to improve the visual quality of fresh-cut 'Rojo Brillante' persimmon, showing a synergic effect with the antioxidant dip (10 g L-1 CA + 10 g L-1 CaCl2). Antioxidant edible coatings were prepared from whey protein isolate (WPI), soy protein isolate (SPI), hydroxylpropyl methylcellulose (HPMC) and apple pectin as the polymeric matrix. All edible coatings were amended with the antioxidant combination selected (10 g L-1 CA + 10 g L-1 CaCl2). All the edible coatings tested proved effective to control enzymatic browning of persimmon slices. However, the samples treated with the HPMC- and pectin- based coatings were scored with a better visual quality that the rest of the treatments. In general, free radical scavenging activity and total carotenoid content increased in late-season persimmons; whereas, processing (cutting and storage at 5 ºC), antioxidant dips, controlled atmosphere storage or edible coatings had no clear effect on nutritional quality (vitamin C, free radical scavenging activity, total phenolic content, and carotenoids) of fresh-cut persimmons.
[ES] El caqui persimmon (Diospyros kaki L.) 'Rojo Brillante' es un cultivar astringente que presenta unas propiedades organolépticas y nutricionales excelentes. En la última década, su cultivo en el área mediterránea de España se ha incrementado de manera exponencial con el desarrollo de la tecnología que permite eliminar la astringencia, manteniendo la firmeza del mismo. Esta nueva forma de presentación, aporta numerosas ventajas, entre la que se incluye la posibilidad de ser comercializado como fruta fresca cortada. Sin embargo, el éxito comercial del producto está limitado por el pardeamiento enzimático, la pérdida de firmeza y al crecimiento microbiano. En este contexto, el objetivo de la Tesis ha sido el desarrollo de caqui 'Rojo Brillante' fresco cortado mediante un enfoque que integra el estudio de las características del producto en el momento del procesado y de distintas tecnologías que mantengan la calidad físico-química, sensorial, nutricional y microbiológica del producto durante un periodo que permita su comercialización. En primer lugar, se evaluó el efecto del estado de madurez (MS) en el momento de recolección, el tiempo de almacenamiento a 15 ºC antes del procesado y la aplicación de diferentes antioxidantes en el pardeamiento enzimático y la calidad sensorial y nutricional del caqui 'Rojo Brillante' cortado y almacenado a 5 ºC. La aplicación de 10 g L-1 de ácido ascórbico (AA) ó 10 g L-1 ácido cítrico (CA) controló el pardeamiento enzimático y mantuvo la calidad visual del caqui por encima del límite de comercialización entre 6 y 8 días de almacenamiento a 5 ºC, dependiendo del MS. Sin embrago, la aplicación de estos antioxidantes redujo de manera significativa la firmeza del fruto respecto al control. La combinación de estos antioxidantes con 10 g L-1 de CaCl2 permitió mantener la firmeza en el mismo rango que las muestras control. En un trabajo posterior, la aplicación de 1-metilciclopropeno (1-MCP) permitió procesar caqui almacenado 45 días a 1 ºC con una buena firmeza comercial y el tratamiento antioxidante (10 g L-1 CA + 10 g L-1 CaCl2) consiguió alcanzar un límite de comercialización del producto de 9 días a 5 ºC. La evaluación de distintas atmósferas controladas en combinación con tratamientos antioxidantes (AA o CA), como paso previo al envasado en atmósfera modificada (MAP) del caqui, mostró como más efectiva en el control del pardeamiento enzimático la atmósfera compuesta por 5 kPa O2 (balance N2). Esta atmósfera mantuvo la calidad visual del caqui cortado dentro del límite de comercialización durante 7-9 días a 5 ºC. Por el contrario, la aplicación de altas concentraciones de CO2 (10 ó 20 kPa) dio lugar a un pardeamiento en ciertas zonas de la pulpa que se conoce como 'internal flesh browning'. Estudios posteriores confirmaron el efecto beneficioso del envasado de caqui cortado y tratado con solución antioxidante (CA-CaCl2) en una MAP activa de 5 kPa O2 en la calidad visual del fruto frente a la aplicación de una MAP pasiva. El desarrollo de recubrimientos comestibles con capacidad antioxidante se realizó mediante la incorporación de antioxidantes (10 g L-1 CA + 10 g L-1 CaCl2) a formulaciones a base de proteína de suero lácteo (WPI), proteína de soja (SPI), hidroxipropilmetilcelulosa (HPMC) y pectina. Todos los recubrimientos fueron efectivos controlando el pardeamiento enzimático del caqui cortado, siendo las muestras recubiertas con HPMC y pectina las mejor evaluadas visualmente. En general, el procesado, la aplicación de antioxidantes, el envasado en atmósferas controladas y los distintos recubrimientos comestibles estudiados, si bien no mostraron un efecto claro en los parámetros de calidad nutricional evaluados, no tuvieron un efecto negativo en los mismos. Por otra parte, los frutos cosechados a final de campaña tuvieron mayor actividad antioxidante y contenido en carotenoides.
[CAT] El caqui persimmon (Diospyros kaki L.) 'Rojo Brillante' és un cultiu astringent que presenta unes propietats organolèptiques i nutricionals excel¿lents. En la última dècada, el seu cultiu en l'àrea mediterrània d'Espanya s'ha incrementat de manera exponencial amb el desenvolupament de la tecnologia que permet eliminar l'astringència, mantenint la fermesa del mateix. Esta nova forma de presentació, aporta un gran nombre d'avantatges, entre els quals s'inclou la possibilitat de comercialitzar-lo com fruita fresca processada. No obstant, l'èxit comercial del producte està limitat per pardetjament enzimàtic, la pèrdua de fermesa i el creixement microbià. L'objectiu de la Tesis ha estat en el desenvolupament de caqui 'Rojo Brillante' tallat en fresc mitjançant un enfocament que integra l'estudi de les característiques del producte en el moment del processat i de diferents tecnologies en el manteniment de la qualitat físico-química, sensorial, nutricional i microbiològica del producte durant un període que permeta la seua comercialització. En primer lloc, es va avaluar l'efecte de l'estat de maduresa (MS) en el moment de recol¿lecció, el temps d'emmagatzemament a 15ºC abans del processat i l'aplicació de diferents tractaments antioxidants en el pardetjament enzimàtic i la qualitat sensorial i nutricional del caqui 'Rojo Brillante' tallat i emmagatzemat a 5 ºC. L'aplicació de 10 g L-1 d'àcid ascòrbic (AA) o 10 g L-1 d'àcid cítric (CA) va controlar el pardetjament enzimàtic i va mantenir la qualitat visual del caqui per damunt del límit de comercialització entre 6-8 dies d'emmagatzemament a 5 ºC, depenent del MS. No obstant, l'aplicació d'antioxidants va reduir de manera significativa la fermesa del fruit comparat amb el control. La combinació d'aquestos antioxidants amb 10 g L-1 de CaCl2 va permetre mantenir la fermesa en el mateix rang que les mostres control. En un treball posterior, l'aplicació de 1-metilciclopropeno (1-MCP) va permetre processar caqui emmagatzemat 45 dies a 1 ºC amb una bona fermesa comercial i a més, el tractament antioxidant (10 g L-1 CA + 10 g L-1 CaCl2) va aconseguir un límit de comercialització del producte tallat de 9 dies a 5 ºC. L'avaluació de diferents atmosferes controlades en combinació amb tractaments antioxidants (AA o CA), com a pas previ a l'envasament en atmosfera modificada (MAP) del caqui 'Rojo Brillante, va mostrar com a més efectiva en el control del pardetjament enzimàtic l'atmosfera composta per 5 kPa O2 (balanç N2). Aquesta atmosfera va mantenir la qualitat visual del caqui tallat dins del límit de comercialització durant 7-9 dies a 5 ºC. Per contra, l'aplicació d'altes concentracions de CO2 (10 ó 20 kPa) va donar lloc a un pardetjament en certes zones de la polpa, el qual és conegut com 'internal flesh browning'. Estudis posteriors van confirmar l'efecte beneficiós de l'envasament de caqui tallat i tractat amb solució antioxidant (CA-CaCl2) en una MAP activa de 5 kPa O2 millorant la qualitat visual de la fruita front a l'aplicació de una MAP passiva. El desenvolupament de recobriments comestibles amb capacitat antioxidant es va realitzar mitjançant la incorporació d'antioxidants (CA-CaCl2) en formulacions a base de proteïna de sèrum làctic (WPI), proteïna de soia (SPI), hidroxipropilmetilcel-lulosa (HPMC) i pectina. Tots els recobriments van ser efectius controlant el pardetjament enzimàtic del caqui tallat. No obstant, les mostres recobertes amb HPMC i pectina van ser millor avaluades visualment que la resta de tractaments. En general, el processat, l'aplicació d'antioxidants, l'envasament en atmosferes controlades i els distints recobriments comestibles estudiats, si bé no van mostrar un efecte clar en els paràmetres de la qualitat nutricional avaluats, no van tindre un efecte negatiu en els mateixos. Per altra banda, els fruits recol¿lectats a final de temporada van tenir major activitat antioxidant i contingut en
Sanchís Soler, E. (2016). Effect of processing on the physicochemical, sensory, nutritional and microbiological quality of fresh-cut 'Rojo Brillante' persimmon [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/62588
TESIS

碩士
國立中興大學
機械工程學系所
104
This study proposes a non-enzymatic glucose sensor fabricated by utilizing photolithography commonly used in the semiconductor industry. First, an ordered array of microhemispherical features was formed on a 6-inch regenerated silicon wafer through photolithography. Next, a thin gold layer was sputtered onto the hemispheres and then gold nanoparticles were uniformly deposited via sol-gel to form a microstructural composite sensing electrode substrate for electrochemistry. It was observed from the results that the effective sensing area of the proposed glucose sensor was 10.2 times greater than a planar gold electrode. Further investigation revealed that the sensor’s linear detection range for glucose was from 55.6 µM to 13.89 mM, with a sensitivity of 749.2 µA·mM-1·cm-2 and a detection limit of 9 µM. In addition, the proposed sensor can also effectively detect changes in glucose levels to an accuracy of ±0.18 mg/dL; far greater than the FDA specification of ±20 mg/dL and ISO15197 specification of ±15 mg/dL. The simple and low-cost manufacturing combined with a high sensitivity, enzyme-free and excellent sensing performace indicates that the proposed non-enzymatic glucose sensor is commercially feasible.

The original objectives of this research project were to: (1) develop immunoassays, photometric sensors, and electrochemical sensors for real-time measurement of progesterone and estradiol in milk, (2) develop biosensors for measurement of caseins in milk, and (3) integrate and adapt these sensor technologies to create an automated electronic sensing system for operation in dairy parlors during milking. The overall direction of research was not changed, although the work was expanded to include other milk components such as urea and lactose. A second generation biosensor for on-line measurement of bovine progesterone was designed and tested. Anti-progesterone antibody was coated on small disks of nitrocellulose membrane, which were inserted in the reaction chamber prior to testing, and a real-time assay was developed. The biosensor was designed using micropumps and valves under computer control, and assayed fluid volumes on the order of 1 ml. An automated sampler was designed to draw a test volume of milk from the long milk tube using a 4-way pinch valve. The system could execute a measurement cycle in about 10 min. Progesterone could be measured at concentrations low enough to distinguish luteal-phase from follicular-phase cows. The potential of the sensor to detect actual ovulatory events was compared with standard methods of estrus detection, including human observation and an activity monitor. The biosensor correctly identified all ovulatory events during its testperiod, but the variability at low progesterone concentrations triggered some false positives. Direct on-line measurement and intelligent interpretation of reproductive hormone profiles offers the potential for substantial improvement in reproductive management. A simple potentiometric method for measurement of milk protein was developed and tested. The method was based on the fact that proteins bind iodine. When proteins are added to a solution of the redox couple iodine/iodide (I-I2), the concentration of free iodine is changed and, as a consequence, the potential between two electrodes immersed in the solution is changed. The method worked well with analytical casein solutions and accurately measured concentrations of analytical caseins added to fresh milk. When tested with actual milk samples, the correlation between the sensor readings and the reference lab results (of both total proteins and casein content) was inferior to that of analytical casein. A number of different technologies were explored for the analysis of milk urea, and a manometric technique was selected for the final design. In the new sensor, urea in the sample was hydrolyzed to ammonium and carbonate by the enzyme urease, and subsequent shaking of the sample with citric acid in a sealed cell allowed urea to be estimated as a change in partial pressure of carbon dioxide. The pressure change in the cell was measured with a miniature piezoresistive pressure sensor, and effects of background dissolved gases and vapor pressures were corrected for by repeating the measurement of pressure developed in the sample without the addition of urease. Results were accurate in the physiological range of milk, the assay was faster than the typical milking period, and no toxic reagents were required. A sampling device was designed and built to passively draw milk from the long milk tube in the parlor. An electrochemical sensor for lactose was developed starting with a three-cascaded-enzyme sensor, evolving into two enzymes and CO2[Fe (CN)6] as a mediator, and then into a microflow injection system using poly-osmium modified screen-printed electrodes. The sensor was designed to serve multiple milking positions, using a manifold valve, a sampling valve, and two pumps. Disposable screen-printed electrodes with enzymatic membranes were used. The sensor was optimized for electrode coating components, flow rate, pH, and sample size, and the results correlated well (r2= 0.967) with known lactose concentrations.

The tomato, a profitable vegetable crop in both the USA and Israel, has benefited significantly from intensive breeding efforts in both countries, and elsewhere (esp. Holland). : Modem hybrids are highly prolific and resistant to a variety of major pests. They produce attractive, firm fruit for both processing and fresh-marketing. In all cases, however, reduction in flavor and aroma have occurred concomitantly with the increase in yield. Sugars-acids ratio dominate fruit taste, whereas aroma volatiles (potent at minute ppb and ppt levels) contribute to the total characteristic tomato flavor. An increase in sugars (1-2%) contributes significantly to tomato fruit taste. However, because of energy reasons, an increase in fruit sugars is immediately compensated for by a decrease in yield. Our main objectives were to: (a) pinpoint and identify the major impact aroma components of fresh tomato; (b) study the genetic and environmental effects on fruit aroma; (c) determine precursors of appealing (flavors) and repelling (off-flavors) aroma compounds in tomato. Addition of saturated salts blocked all enzymatic activities prior to isolation of volatiles by dynamic and static headspace, using solvent assisted flavor evaporation (SAFE) and solid phase micro-extraction (SPME) from highly favored (FA-612 and FA-624) and less preferred (R 144 and R 175) tomato genotypes. Impact aroma components were determined by gas chromatography-olfactometry (GC-O), gas chromatography-mass spectrometry (GC- MS) and aroma extract dilution analysis (AEDA). The potent odorant (Z)-1,5-octadien-3-one, was identified for the first time in fresh tomato. From the ca. 400 volatile compounds in the headspace of fresh tomato, the following compounds are proposed to be impact aroma compounds: (Z)-3-hexenal, hexanal, 1-penten-3-one, 2-phenylethanol, (E)-2-hexenal, phenyl acetaldehyde, b-ionone, b-damascenone, 4-hydroxy-2,5-dimethyl-3-(2H)-furanone (FuraneolR), (Z)-l,5-octadien-3-one, methional, 1-octen-3-one, guaiacol, (E,E)- and (E,Z)- 2,4-decadienal and trans- and cis-4,5-EPOXY -(E)-2-decenal. This confirms the initial hypothesis that only a small number of volatiles actually contribute to the sensation of fruit aroma. Tomato matrix significantly affected the volatility of certain impact aroma components and thus led to the conclusion that direct analysis of molecules in the headspace . may best represent access of tomato volatiles to the olfactory receptors. Significant differences in certain odorants were found between preferred and less-preferred cultivars. Higher consumer preference was correlated with higher concentrations of the following odorants: l-penten-3-one, (Z)-3-hexenal, (E,E)- and (E,Z)-2,4-decadienal and especially Furaneol, whereas lower consumer preference was associated with higher concentrations of methional, 3-methylbutyric acid, phenylacetaldehyde, 2-phenylethanol, and 2-isobutylthiazole. Among environmental factors (salinity, N source, growth temperature), temperature had significant effects on the content of selected aroma compounds (e.g., 3-methylbutanal, 1- penten-3-one, hexanal, (Z)-3-hexenal, (E)-2-hexenal, 2-isobutylthiazole, 6-methyl-5-hepten- 2-one, 1-octen-3-one, methional, 2-phenylethanal, phenyl acetaldehyde, and eugenol) in fresh tomatoes. Salt stress (20 mM NaCl) increased the content of odorants such as (Z)-3-hexenal, 2-phenylethanol and 3-methylbutanal in the R-144 cultivar whereas salinity had minor effects on 1-pentene-3-one, 2-isobutylthiazole and b-ionone. This fundamental knowledge obtained by comprehensive investigation, using modem chemical, sensory and agrotechnical methodology will assist future attempts to genetically modify the concentrations of key odorants in fresh tomatoes, and thus keep the tomato production of Israel and the USA competitive on the world market.