Mediated microbial sensors utilizing two different yeast isolates (SPT1 and SPT2) were developed for the estimation of biochemical oxygen demand (BOD). Measurements of glucose/glutamic acid (GGA) standard solution with potassium ferricyanide mediation resulted in linear ranges extending from the detection limits (i.e. 2 and 5 ppm BOD) to 100 and 200 ppm BOD for the SPT1- and SPT2-based sensors, respectively. The standard error of the mean (SEW for 10 ppm. BOD measurements was 10.1% (SPT1) and 3.9% (SPT2). Response reproducibility had 10.6% error between three identically prepared SPT1 sensors. Response times for concentrations of 20 ppm BOD were within 10 minutes. For pulp mill effluent, the detection limits were 2 (SPT1) and 1 (SPT2) ppm BOD, with SEMs of 3.6% and 14.3% for the SPT1 and SPT2 sensors, respectively. Based on the results obtained in this study, it is concluded that SPT2 is the more suitable biocatalyst for pulp mill wastewater analysis. / While 18S rRNA gene sequence analyses, including BLAST homology searches, have suggested that isolate SPT1 is a close relative of Candida sojae (99.8% homology), no close matches have been found for isolate SPT2. The closest match for SPT2 was to Candida krusei (76.0% homology). Evidence from biochemical tests, fatty acid analysis, and 18S rRNA gene sequence analyses, indicates that isolate SPT2 is a novel yeast species.

<p>As an increasingly prevalent chronic disease, diabetes represents one of the fastest growing health burdens to both the developed and developing world. In an effort to improve the management and treatment of diabetes, implantable sensors that continuously monitor glucose levels have become popular alternatives to patient-administered finger prick measurements of blood glucose. However, following implantation, the performance of these implants suffers from inaccurate and erratic readings that compromise their useful lives. As a result, implantable glucose sensors remain limited as a platform for the reliable management of diabetes. While the interaction between the sensor and its surrounding tissue has been posited as a culprit for erroneous in vivo sensor performance, there remains little evidence to support that theory.</p><p>This dissertation describes the effects that implant-associated tissue reactions have on implantable sensor function. Since tissue response to an implant changes over time, the overall effect of these tissue reactions is broken into two temporal phases: (1) the phase of weeks to months following implantation when a mature foreign body capsule is present around the sensor and (2) the phase of days to weeks immediately following sensor implantation when a provisional matrix of proteins and inflammatory cells envelops the sensor.</p><p>Late stage sensor responses to implantation are marked by both an attenuated sensor signal and a significant time lag relative to blood glucose readings. For this later stage of sensor response, a computational model of glucose transport through the interstitial space and foreign body capsule was derived and implemented. Utilizing physiologically relevant parameters, the model was used to mechanistically study how each constituent part of the capsular tissue could affect sensor response with respect to signal attenuation and lag. Each parameter was then analyzed using logarithmic sensitivity analysis to study the effects of different transport variables on both lag and attenuation. Results identified capsule thickness as the strongest determinant of sensor time lag, while subcutaneous vessel density and capsule porosity had the largest effects on attenuation of the sensor signal.</p><p>For the phase of early stage tissue response, human whole blood was used as a simple ex vivo experimental system. The impacts of protein accumulation at the sensor surface (biofouling effects) and cellular consumption of glucose in both the biofouling layer and in the bulk (metabolic effects) on sensor response were assessed. Medtronic Minimed SofSensor glucose sensors were incubated in whole blood, plasma diluted whole blood, and cell-free platelet poor plasma (PPP) to analyze the effects of different blood constituents on sensor function. Experimental conditions were then simulated using MATLAB to predict the relative impacts of biofouling and metabolic effects on the observed sensor responses. It was found that the physical barrier to glucose transport presented by protein biofouling did not hinder glucose movement to the sensor surface. Instead, glucose consumption by inflammatory cells was identified as the major culprit for generating poor sensor performance immediately following implantation.</p><p>Lastly, a novel, biomimetic construct was designed to mimic the in vivo 3D cellular setting around the sensor for the focused in vitro investigation of early stage effects of implantation on glucose sensor performance. Results with this construct demonstrate similar trends in sensor signal decline to the ex vivo cases described above, suggesting this construct could be used as an in vitro platform for assessing implantable glucose sensor performance.</p><p>In total, it may be concluded from this dissertation that instead of sensors "failing" in vivo, as is often reported, that different physiological factors mediate long term sensor function by altering the environment around the implant. For times immediately following implantation, sensor signals are mediated by the presence of inflammatory macrophages adhered on the surface. However, at longer times post-implantation, sensor signals are mediated not by the consumptive capacity of macrophages, but instead by the subcutaneous vessel density surrounding the sensor as well as the porosity and thickness of the foreign body capsule itself. Taken in concert, the results of this dissertation provide a temporal framework for outlining the effects of tissue response on sensor performance, hopefully informing more biocompatible glucose sensor designs in the future.</p> / Dissertation

Recently a whole cell based biosensor has been developed in our laboratory that consists of a monolayer of human umbilical vein endothelial cells (HUVECs) on the asymmetric cellulose triacetate (CTA) membrane of an ion selective electrode (ISE). When a confluent cell monolayer is formed across the membrane, response from the sensor is inhibited due to inhibited ion transport across the membrane. When the cell based biosensor is exposed to permeability modifying agents, the permeability across the cell monolayer is altered facilitating more ion transport and as a result the response from the sensor increases. This sensor response can be related to the concentration of these agents. One objective of this research was to investigate the ability of the sensor to detect a physiological toxin, alpha toxin from Staphylococcus aureus. Studies demonstrated that the biosensor can detect 0.1ng/ml alpha toxin. Considering the fact that the concentration of this toxin is 100-250 ng/ml in whole blood in humans, this biosensor has the ability to act as the diagnostic tool for staphylococcal diseases. Another part of this research was to investigate the ability of the biosensor to measure angiogenesis by measuring the changes in permeability induced by cytokines such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and tumor necrosis factor andamp;aacute; (TNF- andamp;aacute;) individually and in combination. The sensor response was then compared with the common in vitro assays for angiogenesis. The study demonstrated that at the concentrations studied the sensor response in the presence of cytokines was much higher than that observed for other angiogenesis assays, thereby bolstering the potential of the biosensor to act as a quick screening tool for angiogenesis. Furthermore, epithelial cell based sensor responses to the same cytokines were compared with the responses from endothelial cell based sensor and the mechanisms behind the increased sensor response were elucidated. Finally, to investigate the ability of the sensor to screen cancer, the biosensor was exposed to the serum from healthy individuals and cancer patients. The results showed that the sensor can distinguish between healthy individuals and cancer patients and the results correlate with the stages of cancer.

Persistent organic pollutants and heavy metals are released into the environment through different anthropogenic processes. They are of concern because they tend to bioaccumulate in the food chain and show adverse health effects ranging from acute to chronic toxicity. These pollutants need therefore to be monitored to conserve the environment. Conventionally, samples are sent to a laboratory for analysis by standard techniques such as chromatography and spectroscopy. Although these conventional techniques display high accuracy and low detection limits, they are expensive, require the use of highly trained personnel and tedious sample preparation. In comparison, electrochemical methods such as biosensors are sensitive, low cost and simple to operate. In this thesis, the determination of selected persistent organic pollutants (polybrominated diphenyl ethers, polybrominated biphenyls and polychlorinated biphenyls) and heavy metals (Cd, Pb and Cu) was achieved by the use of amperometric inhibition biosensor based on horseradish peroxidase (HRP) immobilized on the surface of platinum-polyaniline modified electrode. Polyaniline (PANI) film was electrochemically deposited on the platinum electrode surface. The film was characterized by cyclic voltammetry and spectrometric techniques. The CV results proved that the PANI was electroactive and exhibited a fast reversible electrochemistry. Characteristic Ultraviolet–Visible and Fourier Transform Infrared features of the polymer film were identified. They revealed that PANI film synthesized in this study is the conductive emeraldine salt. Horseradish peroxidase based biosensor was constructed by electrostatic attachment of the enzyme onto Pt-PANI electrode surface. Spectrometric and cyclic voltammetric results indicated that the immobilized HRP retained its bioelectrocatalytic activity towards the reduction of hydrogen peroxide. The Pt/PANI/HRP biosensor showed a linear response over a concentration range of 0.05 to 3.17 mM with a detection limit of 36.8 nM. Apparent Michaelis- Menten constant ( app M K ) was calculated as 1.04 mM. This implied that the HRP biosensor had a high affinity for H2O2. Furthermore, the fabricated biosensor showed high sensitivity, good reproducibility, repeatability and long-term stability. The Pt/PANI/HRP biosensor was applied to the determination of selected persistent organic pollutants and heavy metals. The latter was found to inhibit the HRP enzyme’s activity. The percentage inhibition of the investigated persistent organic pollutants decreases in the following order: 2,2´4,4´,6-pentabrominated diphenyl ether> 2-brominated biphenyl> 2-chlorinated biphenyl> 2,2´,4,5,5´-pentachlorinated biphenyl> 2,4,4´-trichlorinated biphenyl. In the case of heavy metals, the degree of inhibition of heavy metals was highest for Cd2+, followed by Cu2+ and then Pb2+. Kinetic study for the amperometric response to H2O2, recorded in the absence and presence of persistent organic pollutants and heavy metals revealed that for polybrominated diphenyl ethers, the inhibition process corresponded to a competitive type whereas for polybrominated biphenyls, polychlorinated biphenyls and heavy metals, it corresponded to the on-competitive type. The biosensor exhibited high sensitivity towards the determination of the metals and persistent organic pollutants as pollutants in real water samples, namely tap water and landfill leachate samples. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2010.

Interactions between single and double stranded oligonucleotides with SiO2 surfaces and the interactions between oligonucleotides and immobilized oligomers have been studied computationally. The oligonucleotide is the 18-base-pair sequence for the survival motor neuron gene SMN1. The oligomer consisted of a 50 unit 2-hydroxyethyl methacrylate (PHEMA) molecule. A linker used to tether the oligonucleotide was either a 10 Å or a 30 Å long succinimdyl 4-[N-maleimidomethyl]cyclohexane-1-caroxylate (sulfo-SMCC-Cn). The surface consisted of a SiO2 crystal that was 50 Å long and 50 Å wide, one unit thick and covered with modified-(3-aminopropyl)trimethoxysilane (m-APTMS) molecules. It was determined that explicit water, sodium counterions and excess salt were necessary to produce computationally stable oligonucleotide structures on surfaces. Artificial partial charges were introduced to the surface, and linkers, oligomers and oligonucleotides were immobilized and studied. The linkers collapsed onto a positive but not onto a negative surface. Oligomers moved closer to the SiO2 surface regardless of the surface charge. Immobilized oligonucleotides tilted significantly from an initial upright position but did not collapse completely onto the surfaces. The interactions between immobilized oligonucleotides and oligomers were examined. The number of oligomers surrounding the oligonucleotide was varied between two and four. Single stranded oligonucleotides were prevented from interacting with the surface as they were inhibited by the presence of oligomers. Double stranded oligonucleotides collapsed onto the surface when only two oligomers were present but remained upright when four oligomers were present. This was due to the four oligomers interacting with one another and effectively shielding the surface. The oligomers interacted with the bases in the single stranded oligonucleotides, making them energetically accessible. Presence of a high density of oligomers prevented the dsDNA from collapsing onto the surface. These results suggest design criteria for preparation of mixed oligonucleotide and oligomer films for use in biosensors.

THESIS SUMMARY:The possibility to print tiny amounts of functional materials in a controllable way is of interest in a large number of applications, for instance in the manufacturing of organic electronics, in the deposition of artificial organs (the so-called organ printing), or in the fabrication of miniaturized devices in areas like biosensing. This has prompted the development of microfabrication techniques capable of depositing such functional materials.The present work is focused on one of these techniques, particularly on a laser-based direct-writing technique called laser-induced forward transfer (LIFT), in which laser pulses are used to deposit material from a donor film to a receptor substrate. This technique is especially interesting due to the possibility that it offers to print droplets of solutions containing functional materials with a high resolution. Moreover, it has additional advantages such as the capability to work in air (at atmospheric pressure and room temperature), the fact that it is a non-contact technique that avoids contamination problems, and the possibility to combine it with laser micromachining.In this work, a complete study of the LIFT of liquid solutions is performed in order to achieve a better understanding of this technique and to find the optimum printing conditions. Such study comprises the analysis of the role that some of the most important experimental parameters have on the printed droplets, as well as the analysis of the mechanisms involved in the ejection and deposition of material through LIFT. This is accomplished by means of both the morphological characterization of the printed droplets at different conditions and the time-resolved imaging of the LIFT process, including the ejection and transport of liquid, as well as the deposition of liquid on the receptor substrate. The present work has provided a complete picture of the LIFT process: the experimental conditions adequate for droplet printing have been set up, and the mechanisms responsible for the generation of droplets have been unveiled. The feasibility of the technique for protein printing has also been demonstrated. Moreover, it has also been found that the preparation of the liquid film in thin form is the major problem that the technique faces. This last aspect has motivated the research of a new technique capable of dispensing with the preparation of the liquid film. This novel technique has been successfully developed and tested in this thesis work.EXTRACTE EN CATALÀ:La present tesi està centrada en una tècnica d'escriptura directa anomenada transferència induïda per làser (amb anglès laser-induced forward transfer, o LIFT), en la qual s'utilitzen polsos làser per dipositar material des d'una pel·lícula donadora a un substrat receptor. Aquesta tècnica és especialment interessant degut a la possibilitat que ofereix per imprimir, amb una gran resolució, gotes de solucions que contenen materials funcionals. A més a més, té avantatges addicionals com ara la capacitat de treballar sense buit i a temperatura ambient, el fet de ser una tècnica que evita problemes de contaminació al no estar en contacte la pel·lícula donadora amb el substrat receptor, i finalment al poder-la combinar amb altres tècniques com ara la micromecanització amb làser.En aquest treball es presenta un estudi complet de la LIFT per tal d'aconseguir-ne una millor comprensió així com trobar les condicions òptimes d'impressió. Els materials dipositats han estat sempre solucions líquides. L'estudi presentat comprèn l'anàlisi dels efectes que alguns dels paràmetres més importants de la tècnica tenen en la morfologia de les gotes transferides, així com l'anàlisi dels mecanismes involucrats en l'ejecció i dipòsit de material a través de la LIFT. Això s'ha aconseguit mitjançant la caracterització morfològica del material transferit a diferents condicions, així com utilitzant l'adquisició ràpida d'imatges del procés d'ejecció de material i de dipòsit. Aquest treball ha permès obtenir una imatge completa del procés de la LIFT: s'han determinat les condicions adequades per la impressió de gotes, i els mecanismes per a la generació de gotes s'han desxifrat. La viabilitat de la tècnica per a la impressió de proteïnes també s'ha demostrat. A més a més, s'ha trobat que la preparació de la pel·lícula líquida per a la LIFT és el principal inconvenient que té aquesta tècnica. Aquest aspecte ha motivat la recerca d'una nova tècnica capaç d'evitar la pel·lícula líquida. Aquesta nova tècnica s'ha desenvolupat i provat satisfactòriament en aquest treball.

Along with an increasing understanding of the harmful effects on the environment of a wide range of pollutants has come the need for more sensitive, faster and less expensive detection methods of identification and quantitation. Many environmental pollutants occur in low levels and often in complex matrices thus analysis can be difficult, time consuming and costly. Because of the availability and easy cultivation of the microorganisms with potentially high specificity, there is considerable interest in the use of living microorganisms as the analytical component (the biocomponent) of sensors for pollutants. While a number of biosensors using bacteria have been developed, yeast has been comparatively rarely used as the biocomponent. Yeast are attractive because they are easy to culture and they are eukaryotes which means their biochemistry is in many respects closer to that of higher organisms. This thesis describes the development of whole cell bioassays that use yeast cells as a sensing element and redox mediators to probe the intracellular redox reactions to monitor the catabolic activity of the yeast resulting from the external substrate, steady-state voltammetry is utilised as the electrochemical detection technique. The isogenic differential enzyme analysis (IDEA) concept of Lincoln Ventures Limited, lead NERF funded research consortium uses bacteria that have been cultured using specific organic pollutants as the carbon source which are the biocomponent in sensors. The use of wild type yeast Arxula adeninivorans that has the ability to use a very wide variety of substrates as sources of carbon and nitrogen was used as an alternative to bacteria to validate the “IDEA” concept. Naphthalene and di-butyl phthalate were chosen as model target contaminant molecules. The performance, detection limits and the usefulness of yeast based biosensor applications for environmental analysis are discussed. This thesis also describes the development and optimisation of a simple, cost effective in vivo estrogens bioassay for the detection of estrogens using either genetically modified or a wild type yeast Saccharomyces cerevisiae. In this study, catabolic repression by glucose was exploited to achieve specificity to estrogens in complex environmental samples that eliminates the requirement for conventional sample preparation. This is the first time that the use of wild type yeast to quantify estrogens has been reported. The attractive features of the bioassay are its use of a non-GMO organism, its speed, its high specificity and sensitivity with a detection limit of 10-15 M. The similarity of binding affinities for major estrogens to those of human estrogens receptors makes this in vivo estrogen bioassay very useful for analytical/screening procedures. The electrochemical detection method also makes it easy to interface with a variety of electronic devices.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph.D.)--Tufts University, 1999. / Adviser: David R. Walt. Submitted to the Dept. of Chemistry. Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 137-158). Also available for download via the World Wide Web; free to University of Oregon users.