This thesis pays attention to electrochemical sensors for the detection of glucose. The theoretical part deals with the technology of thick film, their application and firing. It is further described electrochemistry, electrodes used in electrochemistry and analytical methods based on electrochemical reaction. End of theoretical part deals with electrochemical biosensors for detection glucose using special electrodes based on enzyme glucose oxidase. In the experimental part is processed amperometric measure for detection glucose using screenprinted sensors S10 and S8 with immobilized enzyme glucose oxidase. Next was studied effect of plasma, dissolved potassium chloride, storage, ascorbic acid and the reproducibility of the sensors.

Electrophysiological biosensors enables a novel way to measure electrical activity of biological structures both in-vitro and in-vivo and represents valuable alternative to current cellular activity measuring methods. Within this work we will be focusing on development of organic semiconductor (PEDOT:PSS) based Organic Electrochemical Transistors (OECTs) and optimization of material printing methods used in their development. These transistors are meant to be able to transfer electrochemical signals within the cell membrane to electrical signal. Such sensors should be used for cytotoxicity testing of chemicals and potential drugs on cardiomyocytes. Main benefits of OECTs are in their higher sensitivity thanks to their ability to locally amplify electric signals, better noise-signal ratio and outstanding biocompatibility. Their development is undemanding and inexpensive due material printing methods and materials processable at room temperatures.

Le facteur de motilité autocrine (AMF) est une cytokine sécrétée par les cellules tumorales qui a été détectée dans le sérum et l'urine de patients cancéreux. Cette enzyme stimule la motilité des cellules cancéreuses in vitro et provoque des métastases in vivo. Elle peut être utilisée comme un biomarqueur métastasique.Dans cette étude, un biocapteur électrochimique sensible et spécifique a été conçu pour la détection et la quantification d'une enzyme modèle de l’AMF humain : la PGI de mammifère. Le biocapteur a été construit par liaison de 6-phosphate-D-fructose (F6P) sur une surface d'or d’électrode fonctionnalisée covalemment par des groupements oxyamine.La reconnaissance entre l’enzyme et le biorécepteur a été quantifiée par spectroscopie d'impédance électrochimique et voltammétrie dans une gamme de 10 fM à 100 nM. La limite de détection mesurée est de 6,6 fM. La sélectivité a été prouvée, ainsi que la reproductibilité. Notre biocapteur est une preuve de concept très prometteuse d'un futur dispositif analytique miniaturisé conçu pour la détection rapide, facile et précis de l'AMF. Il pourrait en outre contribuer à valider l'AMF en tant que nouveau biomarqueur du cancer métastatique.Afin d’étudier les interactions mises en jeu dans la reconnaissance entre l’enzyme et le biorécepteur, des études de mécanique moléculaire polarisable via le champ de forces SIBFA ont été réalisées. SIBFA est un champ de forces de seconde génération basé sur les résultats des décompositions ab-initio de l’énergie d’interaction et inclut donc la polarisation mais aussi l’énergie de transfert de charge.Pour cette étude nous avons mis en place deux modèles d’AMF pour SIBFA, une forme entière et une forme réduite, et nous avons construit un mime du biocapteur pour SIBFA. Pour cela, il a fallu concevoir et calibrer chaque fragment nécessaire à l’élaboration du mime. Ensuite différentes minimisations d’énergie ont été réalisées, en prenant en compte ou non la solvatation, puis des études sur les interactions mises en jeu ont été effectuées. / Autocrine motility factor (AMF) is a cytokine secreted by tumor cells that could be detected in the serum and the urine of cancer patients. This enzyme stimulates tumor cells motility in vitro and causes metastasis in vivo. It can be used as a biomarker of metastasis.In this study, a sensitive and specific electrochemical biosensor was designed for the detection and quantitation of a model of the human enzyme AMF: the mammalian PGI. The biosensor was constructed by covalently binding D-fructose 6-phosphate (F6P) on the oxyamine functionalized surface of a gold electrode.Recognition between the enzyme and the bioreceptor was quantified by electrochemical impedance spectroscopy and voltammetry in the range of 10 fM to 100 nM. The measured detection limit was 6.6 fM. Selectivity and reproducibility were also proven. Our biosensor is a promising proof of concept for the design of a future miniaturized analytical device for fast, easy and accurate detection of AMF. It could also help validate the AMF as a new biomarker of metastatic cancer.To study the interactions involved in the recognition process between the enzyme and the bioreceptor, we performed polarizable molecular mechanic studies using the force field SIBFA. SIBFA is a second-generation force field based on the results of ab- initio decomposition energy of interaction and therefore includes not only the polarization but also the charge transfer energy.For this study we have developed two models of AMF for SIBFA, an entire form and a reduced form, and we built a mime of the biosensor for SIBFA. For this, it was necessary to design and calibrate each fragment essential for the development of the mime. Then, different energy minimizations were carried out, some of which taking into account solvation parameters. Studies of interactions between the mime and the AMF model are being carried out.

La conception et le développement de biocapteurs pour la détection directe de biomarqueurs dans un échantillon de fluide biologique constituent un véritable défi dans la recherche pour leur application dans le système de point de soins en diagnostic. Mon projet est axé sur l'élaboration d'une nouvelle plateforme de décomposition biologique composée de graphène qui présente une surface électroactive et spécifique élevée permettant de greffer de nombreux biomolécules avec des métalloporphyrines en tant que nouveau marqueur redox pour suivre le processus de reconnaissance biologique obtenu dans le cas d'un capteur d'ADN. Le concept sera de démontrer que les métalloporphyrines où le potentiel d'oxydo-réduction pourrait être contrôlé par la nature des ions métalliques pourraient être appliquées dans une plate-forme de biocapteurs électrochimique multidétection. L'association de la métalloporphyrine avec le graphène offrira de nombreux avantages, comme la promotion de la réaction de transfert d'électrons et ayant une grande surface pour l'immobilisation des protéines. La détection de la cible présente sera réalisée suite à l'activité redox des métalloporphyrines greffées à la surface. Nous avons déjà synthétisé différentes métalloporphyrines modifiées et caractérisées par des techniques habituelles de spectrométrie telles que la RMN, les UV et la MS. Le biocapteur d'ADN a été construit par le composite de graphène et métalloporphyrine et sonde d'ADN comme biorécepteur suivant l'approche d'attachement covalent et l'optimisation de la construction de biocapteur pour améliorer la stabilité et la reproductibilité. Et nous avons déjà fait une détection de l'ADN cible par diverses méthodes électrochimiques, y compris la voltamétrie cyclique, la voltampérométrie à onde carrée et la spectroscopie d'impédance. Cette stratégie a été réalisée d'abord avec la porphyrine Mn insérée puis généralisée à divers complexes métalliques pour une approche multidétection. Parallèlement, les nanomatériaux hybrides combinant les caractéristiques du graphène (haute conductivité et grande surface spécifique) et des porphyrines (propriétés physiques et chimiques et capacité de transfert d'électrons), tels que la tétraphénylporphyrine portant un et quatre groupes carboxyliques nommés H₂TPP-nCP (n = 1, 4) et la tétraphénylporphyrine incorporant du manganèse (III) (MnTPP-1CP) ont été synthétisés avec succès. L'interaction entre H₂TPP-nCP (n = 1, 4) et l'oxyde de graphène chimiquement réduit (CRGO) a été étudiée dans mon travail de thèse. Pour étudier les propriétés caractéristiques des nanomatériaux préparés, la microscopie électronique à balayage (MEB), la microscopie à force atomique (AFM), les spectres UV-visible, les spectres infrarouges à transformée de Fourier (FT-IR) et les spectres de photoélectrons X. Afin d'éliminer l'absorption non spécifique, la modification de l'électrode par le PEA a également été appliquée et obtenir une excellente application pour la détection de la cible d'ADN. / The design and development of biosensors for direct detection of biomarkers in biological fluid sample is real challenge in research for their application in point of care system in diagnostic. My project is focus on the elaboration of a new platform for biological detaction composed of graphene which exhibits a high electroactive and specific surface suitable for grafting numerous biomoleculer with metalloporphyrins as a novel redox marker for following biological recognition process obtained in the case of DNA sensor. The concept will be to demonstrate that the metaloporphyrines where there redox potential could be controlled by the nature of metal ions could be applied in multidetection electrochemical biosensor platform. The association of metalloporphyrin with graphene will offer numerous advantages, as promoting electron transfer reaction and having a large surface for protein immobilization. The detection of target present will be performed following redox activity of the metalloporphyrins grafted on the surface. We have already synthesized various modified metalloporphyrins and characterizatized by usual spectrometry techniques such as NMR, UV and MS. The DNA biosensor has been constructed by the composite of graphene and metalloporphyrin and DNA probe as bioreceptor following covalent attachment approach and optimization of the biosensor construction for improving stability and reproducibility. And we have already done some detection of DNA Target by various electrochemical methods including cyclic voltammetry, square wave voltammetry and impedance spectroscopy. This strategy was performed firstly with on Mn inserted porphyrin and then generalized to various metal complex for multidetection approach. Meanwhile, the hybrid nanomaterials which combined the features of both graphene (high conductivity and large specific surface area) and porphyrins (physical and chemical properties and electron transfer ability), such as tetraphenylporphyrin bearing one and four carboxylic group named H₂TPP-nCP(n=1, 4) and tetraphenylporphyrin incorporating manganese (III) (MnTPP-1CP) were successfully synthesized. The interaction modes between H₂TPP-nCP(n=1, 4) and chemically reduced graphene oxide (CRGO) was studied in my thesis work. To investigate the characteristic properties of as-prepared nanomaterials, scanning electron microscopy (SEM), atomic force microscopy (AFM), UV-visible spectra, Fourier Transform infrared spectra (FT-IR), X-ray photoelectron spectra (XPS). In order to eliminate the non-specific absorption, the modification of the electrode by monolayer PEA was also applied and obtain excellent application for the detection of DNA target.

Philosophiae Doctor - PhD / Tuberculosis (TB) remains a global epidemic despite the fact that treatment has been available since the 1950’s. This disease is highly contagious and spreads via transmission of the Mycobacterium Tuberculosis (MTB) tubercle via coughing, sneezing and spitting. The disease has various side effects including weight loss, fatigue and even death. To date no cure has been found for TB and thus optimisation of treatment is a constant focus in health related research. TB is highly prevalent in South Africa due to the increased level of patients who are co-infected with HIV. Treatment for TB consists of first line drugs including isoniazid (INH), ethambutol (ETH), pyrazinamide (PYR) and rifampicin (RIF). These drugs are highly effective but also produce many adverse drug reactions (ADR’s) over the 6-month course of treatment. These reactions lead to patients not completing the course, losing quality of life and ultimately adding to the development of drug resistant strains of TB. A method of minimising these ADR’s is the development of a phenotype sensor, which is able to determine the metabolic profile of patients. Metabolic profiles play a huge role in the efficacy of treatment by tailoring treatment in order for patients to stay within the therapeutic range of treatment. This would in turn minimise both toxicity and ineffective treatment. Various methods for the quantification of drugs have been developed such as high performance liquid chromatography (HPLC), mass spectrometry (MS) and ultra-violet visible spectroscopy (UV-vis). / 2023-12-01

In the past decade, the use of nanotechnology as a tool to develop and fabricate new structures and devices for biological sensing and energy applications has become increasingly widespread. In this work, a systematic study has been performed on one-dimensional nanomaterials, with a focus on the development of miniaturized devices with a "bottom up" approach. First, members of the nano - carbon family are utilized for biosensing applications; in particular, carbon nanotubes as well as nitrogen - doped and boron - doped nanocrystalline diamond (NCD) films. These carbon - based materials possess several unique electrochemical properties over other conductive materials which make them suitable for biosensing applications. Single walled carbon nanotubes were deposited on a glass carbon electrode and modified for the detection of Salmonella DNA hybridization. Electrochemical impedance spectroscopy (EIS) was used as the method of detection and a detection limit of 10-9 M was achieved. Nanocrystalline diamond was grown using a microwave enhanced plasma chemical vapor deposition method. The diamond electrodes were doped with either boron or nitrogen to provide substrates and characterization was performed using scanning electron microscopy, atomic force microscopy, Raman spectroscopy, Fourier transform infrared spectroscopy, UV-vis spectroscopy, as well as by electrochemical methods. Modified boron - doped NCD was able to detect Salmonella DNA hybridization via EIS and fluorescent microscopy. The detection limit for these genosensors was found to be 0.4 micrometer complementary DNA. Boron - doped and nitrogen - incorporated nanocrystalline diamond also served as functionalized electrodes for lactic acid detection. It was found that the boron - doped electrodes could detect 0.5 mM lactic acid in a phosphate buffer solution. Second, bismuth antimony nanowires were grown in an anodized alumina template for the fabrication of a thermoelectric cooling device. Bismuth antimony nanowires were chosen due to their high thermoelectric efficiency compared to their bulk material counterpart. The development of a successful anodized template was achieved and EIS was used to diagnose the optimal etch parameters of the barrier oxide layer for nanowire growth. Bismuth antimony nanowires were grown directly on a silicon substrate and a thermoelectric cooling device was fabricated. The nanowires exhibited a thermoelectric efficiency of 0.18 at room temperature.

No description available.

abstract: Proteins are, arguably, the most complicated molecular machines found in nature. From the receptor proteins that decorate the exterior of cell membranes to enzymes that catalyze the slowest of chemical reactions, proteins perform a wide variety of essential biological functions. A reductionist view of proteins as a macromolecular group, however, may hold that they simply interact with other chemical species. Notably, proteins interact with other proteins, other biological macromolecules, small molecules, and ions. This in turn makes proteins uniquely qualified for use technological use as sensors of said chemical species (biosensors). Several methods have been developed to convert proteins into biosensors. Many of these techniques take advantage of fluorescence spectroscopy because it is a fast, non-invasive, non-destructive and highly sensitive method that also allows for spatiotemporal control. This, however, requires that first a fluorophore be added to a target protein. Several methods for achieving this have been developed from large, genetically encoded autofluorescent protein tags, to labeling with small molecule fluorophores using bioorthogonal chemical handles, to genetically encoded fluorescent non-canonical amino acids (fNCAA). In recent years, the fNCAA, L-(7-hydroxycoumarin-4yl)ethylglycine (7-HCAA) has been used in to develop several types of biosensors. The dissertation I present here specifically addresses the use of the fNCAA L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) in protein-based biosensors. I demonstrate 7-HCAA’s ability to act as a Förster resonance energy transfer (FRET) acceptor with tryptophan as the FRET donor in a single protein containing multiple tryptophans. I the describe efforts to elucidate—through both spectroscopic and structural characterization—interactions within a 7-HCAA containing protein that governs 7-HCAA fluorescence. Finally, I present a top-down computational design strategy for incorporating 7-HCAA into proteins that takes advantage of previously described interactions. These reports show the applicability of 7-HCAA and the wider class of fNCAAs as a whole for their use of rationally designed biosensors. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

Identifying and detecting health hazards in food products, especially contaminants and toxic substances such as allergens, food toxins and agricultural residues from pesticides, remains a challenge. Increasing demand for food products and growing health consciousness necessitate rapid and accurate measurements which can be easily conducted on-site without long measurement times and high costs. Due to their ease of use, accuracy sample preparation and rapidity, biosensors have started to outcompete time-consuming lab-scale analytical devices. However, as the use of biosensors increase, a concern of the amount of plastics and synthetic polymers used in the fabrication of these biosensors rises. In this dissertation, new ways to create biodegradable and eco-friendly plant-based SERS biosensor platforms from corn protein, zein, are presented. Its higher hydrophobicity and film forming capability make zein a very suitable biopolymer for fabricating biosensors. In the first part of this dissertation, chemical crosslinking was tested to improve the surface hydrophobicity, surface roughness (using AFM), mechanical properties, kinetics of gelation and film formation of zein films, and as a result zein-film based SERS platforms with fewer defects could be fabricated. In the second part, the detection sensitivity of the zein film-based SERS platforms was increased with metallic nanoparticle decoration (gold, silver or silver-shelled-gold). The addition of all three types of nanoparticles significantly increased the SERS enhancement factors of the platforms, with silver-shelled-gold nanoparticles giving the highest enhancement factor of 10⁵. In the last part of this thesis, a novel approach was tested, where electrospun zein nanofibers decorated with metallic nanoparticles were used as a SERS biosensor platform. Due to their higher surface area-to-volume ratios, electrospun zein nanofibers gave a higher SERS enhancement factor (10⁶). This enhancement factor enabled the detection of acrylamide, a food carcinogen, with a 10⁴ times lower detection limit than nanophotonic based nanoimprinted zein, acrylamide sensor platform. Overall, this dissertation successfully shows the fabrication of biodegradable and eco-friendly SERS sensor platforms that have comparable detection sensitivities to those of non-biodegradable ones.

<p>Two thirds of patients with diabetes avoid regularly monitoring their blood glucose levels because of the painful and invasive nature of current blood glucose detection. As an alternative to blood sample collection, exhaled breath condensate (EBC) has emerged as a promising non-invasive sample from which to monitor glucose levels. However, the inconsistency in the methods used to collect EBC significantly impacts the reliability of reported analyte concentrations in EBC. Furthermore, this dilute sample matrix requires a highly sensitive glucose biosensor to enable robust and accurate glucose detection at the point-of-care. Together, a reliable collection method and sensitive detection system can enable accurate modeling of glucose transport from blood to breath that is reflective of airway glucose homeostasis.</p> <p> I address this research gap by simultaneously designing a standardized EBC collection method that allows for separation of dead space and alveolar air and developing a competitive fluorescent biosensor that can resolve micromolar glucose concentrations changes. First, I develop a low-cost, automated condenser that selectively collects exhaled breath that has been exchanged with lung fluid based on the detection of higher breath temperatures that are characteristic of the lower respiratory regions. Using this device, I investigate the relationship between blood and EBC glucose in diabetic and normoglycemic human subjects. Next, I engineer the exquisitely sensitive <i>E. coli</i> glucose binding protein (GBP) with a chemo-enzymatic tag to selectively conjugate it to highly photostable quantum dots (QDs). Finally, I take advantage of the competitive binding of glucose (K_D=0.35 µM) and galactose (K_D=1.4 µM) to GBP to develop a fluorescent glucose biosensor using the GBP-QD conjugate.</p>