Author: Elizabeth Schneider
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ISBN:
Category :
Languages : en
Pages : 214
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Cytochrome P450s (CYPs) are a family of enzymes implicated in the metabolism of drugs in the body. Consequently, P450 reactions are of high interest to the pharmaceutical industry, where lead compounds in drug development are screened as potential substrates of CYPs. The P450 reaction involves electron transfer to an iron heme via NADPH and the electron transfer partner enzyme P450 reductase (CPR). By immobilizing CYPs on an electrode however, NADPH and CPR are potentially no longer needed and the immobilized CYP can act as a biosensor by accepting electrons directly from the electrode. Such a biosensor could be used as an initial screening tool for CYP reactivity of pharmaceuticals in development. In this study, the drug-metabolizing enzyme CYP 2C9 was immobilized to a self-assembled monolayer (SAM) on a gold electrode in three different orientations to investigate the effect that orientation has on the direct electrochemistry of CYP and to evaluate oriented attachment of CYP to an electrode as a biosensor design. Three attachment methods were investigated: random attachment via amine coupling to a carboxy-terminated SAM, oriented attachment via C-terminal His-tag coupling to a Ni-NTA-functionalized SAM, and oriented attachment via maleimide/thiol coupling to a maleimide-functionalized SAM. Three 2C9 mutants (R125C, R132C, and K432C) were developed with a single cysteine mutation at the binding site for CPR on the side of the enzyme closest to the heme; attachment of these mutants to a gold electrode via maleimide/thiol coupling would orient the enzyme such that electron transfer occurs on the electrode in the same orientation that it does in vivo with CPR. Therefore, we expected oriented attachment via maleimide/thiol coupling to produce the most electroactive CYP biosensor. Electrochemical analysis and surface characterization of the SAMs on gold electrodes confirmed that electron transfer occurs through the SAMs, and activity assays of the 2C9 electrodes confirmed that wild-type 2C9 and the single Cys mutants R125C, R132C, and K432C were immobilized to the gold electrode via all three attachment methods. Cyclic voltammetry of the 2C9 electrodes revealed however, that direct electron transfer to 2C9 was not possible for all three attachment methods. Similar redox processes were observed for both the 2C9 electrodes and no-enzyme electrodes modified only with SAMs, suggesting that the redox process observed on the 2C9 electrodes is related to the underlying SAM. Thus, we were unable to make any conclusions regarding the effectiveness of oriented attachment in creating a 2C9 biosensor. However, to our knowledge, there are no examples in the literature of the oriented attachment of 2C9 to an electrode via coupling of an engineered cysteine to a maleimide-functionalized SAM on gold and therefore this study represents the first attempt towards such an electrode system.
Author: Cherie Tollemache
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ISBN:
Category : Biosensors
Languages : en
Pages : 305
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It is important to develop highly selective and sensitive biosensors that can reproducibly detect a target analyte in a rapid and cost-effective way. The creation of alkanthiolate self-assembled monolayers (SAMs) is a common approach to functionalise gold electrode materials for biosensor applications. This thesis evaluates a SAM-based electrochemical biosensor design proposed by a small engineering firm (the Company) and optimise the surface functionalisation methodology and bioassay conditions towards detection of progesterone for agricultural applications. Chapter 1 presents a general introduction to the field of biosensors and the design considerations that are essential to the functioning of biosensors. Recent examples of approaches to detecting analytes in low concentration in complex biological samples using elegant biosensor designs are surveyed. The use of alkanethiolate self-assembled monolayers as a method for surface functionalisation is introduced. The impact of the methodology for SAM formation on the properties and function of the modified surface is examined. The use of this surface functionalisation strategy to create electrochemical biosensors is discussed and the objectives of this thesis are outlined. Chapter 2 describes the methodology used for the experimental work performed in this thesis. The surface characterisation techniques used in the thesis are outlined and the relevance of these techniques for determining the reproducibility, composition and quality of mixed alkanthiolate self-assembled monolayers as functional surfaces for biosensor applications are evaluated. Chapter 3 describes a series of experimental measurements designed to evaluate the sensor electrode system designed by the Company. The experiments perfomed examined the SAM deposition methodology effects on the biosensor response including experimentation with diluent chain length and ratio of adsorbates in solution. The reaction conditions for attachment of DNA probes to the SAM coated surfaces were explored and experiments regarding calibration of the sensor response and detection of progesterone in buffer were performed. It was found that the sensor design provided measurable signals that were affected by changes in the SAM deposition conditions and attachment chemistry method, however, there was an unacceptable level of variation in sensor response in every experiment. A series of issues were identified in both the biosensor architecture and the surface chemistry which led to the irreproducible bioassay results reported. These limitations in sensor design are discussed and suggested improvements are outlined. Chapter 4 describes experiments designed to examine some of the hypothesised surface chemistry contributions to the irreproducibility of sensor response discovered in Chapter 3. The limitations in device architecture were mitigated by switching to an alternative electrode design and the methodology of mixed SAM deposition and subsequent reaction chemistry is examined in detail. A systematic study of the electrochemical properties of mixed SAMs incorporating carboxylic acid or azide functional groups mixed with hydroxyl or methyl terminated diluents with different chain lengths is reported. The ratio of the functional to diluent molecule is varied and short and longer deposition times are compared. The amide bond coupling to mixed SAMs with carboxylic acid functionality and click chemistry reactions with azide terminated mixed SAMs were examined by the attachment of ferrocene derivatives to the SAMs. This was also used as a means of evaluating the compositional reproducibility of the mixed SAMs and the reproducibility of surface reactions for these different systems. Chapter 5 presents preliminary results for a novel methodology for creating mixed SAMs from thioacetyl-protected adsorbate species that occurs on a significantly faster time-scale than the methods used in Chapters 3 and 4. The purpose of this work was to evaluate this method compared to the more conventional methodology as a possible approach for creating mixed SAM modified surfaces of a controllable composition and quality for commercial biosensor applications. The new method involves coadsorptiopn of alkanethiolates generated in situ by deprotection of thioaceytl-protected adsorbates under basic conditions. This proof-of concept study examines some of the factors that influence the SAM formation under these new deposition conditions including solvent effects and the use of adsorbate molecules with hydrophilic and hydrophobic terminal groups. Evaluation of the reproducibility of this method is performed in two ways, with one researcher performing the experiments multiple times and a comparison between two different researchers performing the experiments. Chapter 6 provides a brief summary of the conclusions determined from the experimental results and literature analysis with emphasis on the broader context of the commercial objectives of this thesis. This chapter also provides a discussion on future experimentation that could be performed to improve the understanding of the mixed SAMs systems studied. Experimentation with SAM deposition methodology and reaction chemistry to further functionalise electrode surfaces towards biosensor applications did not lead to discovery of a system that is sufficiently reproducible for the intended application. The use of SAMs on gold prepared by methods not explored in this thesis may provide the reproducibility in electrochemical properties that is required for electrochemical biosensors. A company aiming to create a biosensor for commercial use would be advised to explore other methods of surface functionalisation or design a bioassay/device architecture that does not rely so heavily on precisely reproducible surface functionalisation.
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Category :
Languages : en
Pages :
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Author: Thomas Wayne Schneider
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Category : Adsorption
Languages : en
Pages : 198
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Author: Jakub Trzebinski
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Category :
Languages : en
Pages :
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Designing a biosensor capable of continuously monitoring blood glucose concentration in people with diabetes has been a major challenge for over three decades. In this work we attempt to develop a novel microspike based minimally invasive biosensor for this purpose. Also, as a part of an ongoing study, we attempt to improve the current design of coil-type implantable biosensors. Microspikes, which are able to painlessly penetrate the skin layer, were fabricated using lithographic techniques and sputtered with gold to serve as an electrode. The biosensor design is based on thiomalic acid self-assembled monolayer (SAM) on which glucose oxidase was immobilised by a simple coupling technique together with a tetrathiafulvalene mediator entrapped in an epoxy-polyurethane permselective membrane. Functional testing revealed that such modified sensors are capable of detecting glucose concentration within the clinically relevant range. This was followed by studying the microspike based biosensors incorporated into the microfluidics platform mimicking the sensor behaviour in interstitial fluid. Data from these experiments revealed that the sensor response is mainly dependent on enzyme kinetics rather than membrane permeability to glucose. In contrast, an attempt to address the reproducibility issues of coil-type biosensors is presented. The hypothesis for this study was that a crosslinked hydrogel would have a sufficiently uniform porosity and hydrophilicity to address the variability in sensor sensitivity. The hydrogel was prepared by crosslinking di-hydroxyethyl methacrylate, hydroxyethyl methacrylate and N-vinyl pyrrolidone with 2.5 mol% ethylene glycol dimethacrylate using the water soluble initiators - ammonium persulphate and sodium metabisulfite under a nitrogen atmosphere. The hydrogel was applied to the sensor by dip coating during polymerisation. Electrochemical measurements revealed that the response characteristics of sensors coated with this membrane are highly consistent. Scanning Electrochemical Microscopy (SECM) was used to spatially resolve glucose diffusion through the membrane by measuring the consequent hydrogen peroxide release and compared with an epoxy- polyurethane membrane.
Author: Dusan Losic
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ISBN:
Category : Gold
Languages : en
Pages : 9
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Author: Lawrence Awa Ticha
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ISBN:
Category : Nano-composites
Languages : en
Pages : 220
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Author: Maria Pilar Calatayud Sanz
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ISBN:
Category :
Languages : en
Pages : 176
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Author: M. Y. Ho
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Category :
Languages : en
Pages :
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Author: Maria Pilar Calatayud Sanz
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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