Tesis sobre el tema "Biosensor"

Thesis (M.S.)--West Virginia University, 2008.
Title from document title page. Document formatted into pages; contains vi, 120 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 91-100).

This work describes the development of mediated amperometric biosensors that are able to monitor the metabolic activity of both single and mixed microbial populations, with applications in toxicity assessment and wastewater treatment plant protection. Biosensor systems have been constructed incorporating either the single-species eubacteria Escherichia coli or Pseudomonas putida, Bioseed®, or a mixture of activated sludge organisms from wastewater treatment plants, as the sensing components immobilised on disposable screen printed electrodes in stirred reaction vials. The biosensor approach is generic allowing for a wide range of microbial cell types to be employed. Appropriate bacterial species can be selected for specific sensor applications in order to confer validity and relevance to the test, hence the biosensor can be tailor-made to assess the toxicity in a particular environment and provide diagnostically valid and relevant results. The biosensors have been used to assess the toxicity of a standard toxicant and toxicant formulations and in blind testing of a range of industrial effluents, in parallel with a number of bioassays including Microtox® and activated sludge respiration inhibition. The biosensor results generally show significant correlation to the appropriate conventional toxicity tests. In this study, an activated sludge based biosensor assay was developed and used to assess the toxicity of industrial process and site effluents with the specific purpose of wastewater treatment plant protection. Data generated compared significantly with those from an activated sludge respiration inhibition test, with added advantages of rapidity, safety and ease of use.

The objective of this research was to develop the initial constituents of a highly scalable and label-free electronic biosensing platform. Current immunoassays are becoming increasingly incapable of taking advantage of the latest advances in disease biomarker identification, hindering their utility in the potential early-stage diagnosis and treatment of many diseases. This is due primarily to their inability to simultaneously detect large numbers of biomarkers. The platform presented here - termed the electronic microplate - embodies a number of qualities necessary for clinical and laboratory relevance as a next-generation biosensing tool. Silicon nanowire (SiNW) sensors were fabricated using a purely top-down process based on those used for non-planar integrated circuits on silicon-on-insulator wafers and characterized in both dry and in biologically relevant ambients. Canonical pH measurements validated the sensing capabilities of the initial SiNW test devices. A low density SiNW array with fluidic wells constituting isolated sensing sites was fabricated using this process and used to differentiate between both cancerous and healthy cells and to capture superparamagnetic particles from solution. Through-silicon vias were then incorporated to create a high density sensor array, which was also characterized in both dry and phosphate buffered saline ambients. The result is the foundation for a platform incorporating versatile label-free detection, high sensor densities, and a separation of the sensing and electronics layers. The electronic microplate described in this work is envisioned as the heart of a next-generation biosensing platform compatible with conventional clinical and laboratory workflows and one capable of fostering the realization of personalized medicine.

Introduction: A biosensor may be described as a sensor incorporating a biological element such as an enzyme, antibody, nucleic acid, microorganism or cell. A biosensor should exhibit both shelf-stability and operation stability. Compatible solutes from hyperthermophilic bacteria, called hypersolutes, are very efficient for the preservation of the performance of a wide variety of biomaterials; ranging from proteins to whole cells and artificial tissues. The overall objectives of this work have been to investigate the application of hypersolutes to enhance the performance of biosensors based on the stabilization properties offered by hypersolutes compounds, particularly with respect to storage and operational lifetime. Materials and Methods: The stabilizing agents considered for this study were firoin, firoin A, ectoine®, hydroxyectoine, diglycerol phosphate (DGP) and potassium mannosyl-lactate (PML), provided either by Bitop AG (Witten, D) or StabVida (Oeiras, P).The following enzymes were selected due to their commercial importance: Glucose oxidase (GOx), alcohol oxidase (AOx), acetylcholinesterase (AchE) and lactate dehydrogenase (LDH). On immunosensors, a model system was first designed using ELISA tests. The influence of hypersolutes was then studied using BIAcore. The antibody test system selected for examination of the effect of stabilizing agents on immunosensor performance was based on an anti-human immunoglobulin G (IgG) primary antibody, grown in goat, and an anti-goat secondary antibody conjugated to horseradish peroxidase enzyme. A model DNA binding system was sought: The poly-A strand was tethered to the sensing surface within the BIAcore system via a biotin-streptavidin linkage whereas the complimentary poly-T strand contained a fluorescent Cy3 label, that offered the possibility to also use more conventional detection techniques to ensure thathybridization between the two complimentary strands had been achieved, as well as add a significant weight to the strand, increasing its visibility on the BIAcore signal.

The silicon based sensor is able to detect part per trillion ionic silver in 0.0098% hydrofluoric acid based on the open circuit potential (OCP) measurement. The OCP jump of 100 ppt ionic silver solution is up to 120 mV. The complex agent can effectively suppress the ionic silver concentration and suppress the OCP signal. The ability of complex agent to suppress the OCP signal depends on the formation constant of the complex with silver. The complex adsorbed on the sensor surface induces a second OCP jump, the height of the second jump depends on the formation constant of the complex. The MINEQL chemical equilibrium modeling program is used to calculate the ionic silver concentration when complex agent presents, a discrepancy is found between the MINEQL simulation result and the OCP signal of the silicon based sensor. The toxicity of ionic silver to C. dubia is studied parallel to the OCP signal of silicon based sensor. Less toxicity is found when the complex agent is present similar to the OCP signal. Another discrepancy is found between the MINEQL simulation and the toxicity test when MINEQL simulation is used to predict and control the ionic silver concentration. The data from both biosensor C. dubia and silicon based sensor support each other and both are not in agreement with MINEQL simulation prediction.

Nanostrukturen haben in den letzten Jahrzehnten durch konsequente Förderung wie der im Jahr 2000 gestarteten National Nanotechnology Initiative der USA oder des deutschen Pendants Aktionsplan Nanotechnologie erhebliches Aufsehen, nicht nur in der Wissenschaft, sondern auch in der technischen und wirtschaftlichen Umsetzung erfahren. In Kombination mit biologischen Systemen, deren Funktionalität sich auf der Größenordnung von Nanometern abspielt, finden nanotechnologische Entwicklungen auf dem Gebiet der Medizin ein großes technisches Anwendungsgebiet. Diese Arbeit widmet sich der Untersuchung und technischen Entwicklung von Siliziumnanodrähten als Sensoren für zukünftige medizinische Anwendungen. Im Gegensatz zu Sensoren die auf dotierten Nanodrähten basieren, wurden hier undotierte Nanodrähte untersucht, die mit geringerem Produktionsaufwand auskommen und mittels Schottky-Barrieren als Feldeffekttransistoren nutzbar sind. Deren Eigenschaften wurden im Hinblick auf pH und Biosensorik theoretisch und experimentell untersucht, sowie technisch in ein lab-on-chip sowie ein kompaktes Multiplexer-Messgerät integriert. In einem zweiten, separaten Teil wurden die Eigenschaften undotierter Nanodrähte für die optische Spektroskopie theoretisch modelliert. Die Inhalte beider Teile werden im folgenden kurz zusammengefasst. Um die elektrischen Sensoreigenschaften der Siliziumnanodrähte zu untersuchen, wurden zunächst Computermodelle der Drähte erstellt, mit deren Hilfe der Elektronentransport in flüssiger Umgebung quantenmechanisch modelliert wurde. Die dafür erstellten Modellvorstellungen waren für die sich daran anschließenden experimentellen Untersuchungen des Rauschverhaltens, der pH-Sensitivität sowie der Biosensoreigenschaften sehr vorteilhaft. Mit Hilfe einer neu entwickelten Messmethode konnte der optimale Arbeitspunkt der Sensoren ermittelt werden, sowie die hohe Sensorqualität mittels einer empirischen mathematischen Beschreibung des zu erwartenden Sensorsignals eingeordnet werden. Weiterhin wurden für die Medizintechnik relevante Messungen von Thrombin durchgeführt. Damit ist für den hier beschriebenen Sensortyp ein proof-of-concept für neuartige medizinische Messelemente gelungen. Um die kleinen Abmessungen der Sensoren darüber hinaus technisch nutzbar zu machen, wurden sie in ein lab-on-chip System integriert, in welchem sie als Sensoren für den pH-Wert sowie die ionische Konzentration in Nanoliter-Tropfen verwendet wurden. Desweiteren wurde in Kooperation mit dem Institut für Aufbau- und Verbindungstechnik ein portables Messgerät entwickelt, welches die parallele Messung mehrerer Nanodrahtsensoren ermöglicht. Im zweiten Teil der Arbeit wird eine theoretische Untersuchung zur Eignung von Silizium-Nanodrähten als Messsonden (Probes) für die optische Spektroskopie vorgestellt. Dazu wurde eine Methode entwickelt mittels derer es möglich ist, Raman und Infrarotspektren von Nanostrukturen mittels Molekulardynamik zu berechnen. Die Methode wurde auf undotierte Silizium-Nanodrähte augewendet und zeigt, dass die Oberflächenbeschaffenheit der Drähte die optischen Spektren entscheidend beeinflusst. Damit konnte die Relevanz von Halbeiter-Nanostrukturen auch für Anwendungen in der optischen Spektroskopie gezeigt werden
Nanostructures have attracted great attention not only in scientific research, but also in engineering applications during the last decades. Especially in combination with biological systems, whose complex function is controlled from nanoscale building blocks, nanotechnological developments find a huge field of applications in the medical sector. This work is dedicated to the functional understanding and technical implementation of silicon nanowires for future medical sensor applications. In contrast to doped silicon nanowire based sensors, this work is focussed on pure, undoped silicon nanowires, which have lower demands on production techniques and use Schottky-barriers as electric field detectors. The pH and biosensing capabilities of such undoped silicon nanowire field effect transistors were investigated theoretically and experimentally and further integrated in a lab-on-a-chip device as well as a small-scale multiplexer measurement device. In a second separate part, the optical sensing properties of undoped silicon nanowires were theoretically modeled. The main contents of both parts are shortly described in the following paragraphs. A multiscale model of silicon nanowire FETs to describe the charge transport in liquid surrounding in a quantum mechanical framework was developed to investigate the sensing properties of the nanowire sensors in general. The model set the basis for the understanding of the subsequent experimental investigations of noise characterization, pH sensitivity and biosensing properties. With the help of a novel gate sweeping measurement method the optimal working point of the sensors was determined and the high sensor quality could be quantified in terms of an empirical mathematical model. The sensor was then used for measurements of medically relevant concentrations of the Thrombin protein, providing a proof-of-concept for medical applications for our newly developed sensor. In order to exploit the small size of our sensors for technical applications we integrated the devices in lab-on-a-chip system with a microfluidic droplet generation module. There they were used to measure the pH and ionic concentration of droplets. Finally a portable multiplex measurement device for silicon nanowire sensors as well as other ion sensitive FETs was developed in cooperation with the IAVT at TU Dresden (Institut für Aufbau- und Verbindungstechnik). The second part of this thesis investigates the usability of silicon nanowires for optical sensor applications from a theoretical point of view. Therefore a method for the extraction of Raman and Infrared spectra from molecular dynamics simulations was developed. The method was applied to undoped silicon nanowires and shows that the surface properties of the nanowires has a significant effect on optical spectra. These results demonstrate the relevance of semiconductor nanostructures for applications in optical spectroscopy

The development of an enzyme biosensor employing a novel functionalised polythiophene matrix is presented. The research upon conducting polymer platforms for biological immobilisation is extensive but by no means exhaustive and therefore this investigation contributes to the field of glucose detection with covalently immobilised glucose oxidase upon novel copolymers of N-succinimido thiophene-3- acetate/3-methylthiophene (STA-MT), trans-3-(3-thienyl) acetic acid/3- methylthiophene (TTA-MT) and N-succinimido trans-3-(3-thienyl) acetate/3- methylthiophene STTA-MT. Polymer characterisation was performed using electrochemical techniques, primarily cyclic voltammetry. The examination of various substituted conducting polymers from the polythiophene family was performed where 3-methylthiophene was selected for copolymerisation due to its low oxidation potential and redox behaviour. Copolymerisation of the novel monomers provided reversible and stable films and succeeded in generating a range of copolymer ratios characterised by cyclic voltammetry with the available binding sites calculated using SEM-EDX. Film morphology and dopant intercalation were investigated, providing supporting evidence for the successful copolymerisation of novel monomers with 3- methylthiophene. Optimisation of the biosensor format was investigated through analysis of film thickness, copolymer ratio and enzyme immobilisation time. A film thickness generated with 50 mC provided a stable film with good response to glucose over a wide range of immobilisation times and was employed for all other biosensor investigations. A 10% functional monomer content provided enhanced current signals upon glucose addition which supported the findings of Kuwahara et al where a 10% thiophene-3-acetic acid content within a thiophene-3-acetic acid/3-methylthiophene copolymer demonstrated enhanced biosensor behaviour. All studies were compared against the thiophene-3-acetic acid and 3-methylthiophene system generated under the optimised conditions of the novel copolymer films. The evaluation of the immobilised glucose oxidase as an amperometric biosensor within a mediated system employing p-benzoquinone was performed generating fast response times within 4 seconds, good stability and excellent current response to glucose between 1.4 and 1.9 mA/cm2 after the addition of 15 mM of glucose. All three novel biosensor systems exhibit good analytical performance demonstrating excellent repeatability typically over 90%, reproducibility of over 80% and shelf-life stability of 85-96% over a seven day analysis and between 50-78% after 1 month.

The concepts of electroneutrality coupling and electron-hopping, which are useful for the incorporation of functional components and transportation of electrons, were applied in this project. Discrete layered structures were fabricated by sequential electropolymerization to modulate the performances of formate biosensors. Different types of layers, with or without enzyme, were successfully grown on the electrode surface. The presence of the enzyme (formate dehydrogenase), co-factor (B-nicotinamide adenine dinucleotide) and an electron mediator in the polypyrrole film was verified by scanning electron microscopy, chronopotentiometry, cyclic voltammetry and amperometric measurements. Monolayer, bilayer and trilayer formate biosensors were successfully fabricated for different analytical purposes. The utilisation of the biosensing membrane for the reliable batch and FIA determination of formate based on a amperometric mode of detection are explored. Electron mediators such as ferrocyanide, Prussian Blue, ferrocene and ferrocene carboxylic acid were incorporated into the polypyrrole film to lower the required applied potential for amperometric sensing and to maintain the conductivity and stability of the polypyrrole backbone. The application of artificial neural networks (ANN) to overcome the problem of reusability and reproducibilty in a nonlinear and complicated dynamic system is also considered. The resulting system was trained with a new neural network based software package, Turbo Neuron, for prediction of the concentration of formate, based on the entire collected data, which contain the history of the detection experiments. The proposed integrated ANN conducting polymer biosensor enables the determination of formate concentration, both online and in real time
Doctor of Philosophy (PhD)

This thesis reports the design and development of the first waveguide surface plasmon resonance (SPR) biosensor for pesticide analysis. The integrated optical format allows the fabrication of a compact sensor that may be connected to optical fibre; necessary steps towards a portable optical biosensor. A major advantage of the integrated optical approach is the possibility of fabricating multiple sensors on one substrate, and hence testing for multiple analytes in one basic assay. The SPR structure incorporates a metal film that may be employed as an electrode to study the electrochemical control of sensing reactions. The performance of such devices requires analysis of the waveguide modes supported by the metal-clad waveguide, of their excitation by an input waveguide and of the resultant power coupled into an output waveguide. For the first time a rigorous numerical waveguide model to study the power transmission of such general multilayer absorbing structures has been developed. The model allows the determination of the modulation in output power of the sensor due to the adsorption of a thin organic layer to the sensor surface, which in turn leads to a measure of sensitivity. Designs for practical, sensitive, waveguide SPR sensors for an aqueous environment, optimised for specific sensing films are reported. The fabrication of gold-coated, potassium ion-exchanged, waveguide SPR sensors in soda-lime and Pyrex glass is reported. Three types of experiment were performed to validate the waveguide model using these devices. The first involved measuring changes in the output power of the sensor as a function of gold film length. The second measured the SPR response of sensors as a function of gold film parameters. Third, the effect on the SPR response of binding a dual layer of biotin-avidin to the sensor surface was observed as a function of gold film thickness. Predictions of the waveguide model were compared to the experimental data. Optimisation of the sensor design through these experimental procedures is also described. The transformation of the basic waveguide SPR sensor into a specific biosensor for the triazine herbicides simazine and atrazine is reported. The assay procedure was based on anti-simazine and anti-atrazine IgG antibodies and their Fab fragments developed by co-workers at GEC Marconi Materials Technology Ltd., UK Chemical modification of the sensor surface was developed by co-workers at the University of Tubingen, Germany, to bind the antibodies to the sensor surface. Laboratory characterisation of the sensor as a simazine sensor was performed and is reported in this thesis. Extended validation identified a detection limit of 0.22µg/l for the herbicide simazine in the aqueous environment. The biosensor gave a significant correlation with HPLC measurements on natural water samples when the cross-reactivity of the sensor with other triazine herbicides was taken into account.

Thesis (M.S.)--West Virginia University, 2004.
Title from document title page. Document formatted into pages; contains viii, 109 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 107-109).

Existe una demanda creciente de dispositivos de análisis multianalito, con aplicaciones potenciales en los campos de la biomedicina y biotecnología, así como en el ámbito industrial y ambiental. Para el desarrollo de estos dispositivos resulta esencial un buen control espacial durante la etapa de inmovilización de las biomoléculas de interés; cada una de ellas debe ser depositada de forma precisa sobre la superficie del sensor (por ejemplo, un transductor amperométrico), evitando solapamientos que puedan comprometer la especificidad del sistema. El objetivo de esta tesis es desarrollar diferentes métodos de patterning para la inmovilización selectiva de biomoléculas. El primer método consiste en la electrodeposición selectiva de nanopartículas de oro biofuncionalizadas para el desarrollo de biochips. Se trata de un método de patterning controlado electroquímicamente, en el que las nanopartículas de oro se modifican en primer lugar recubriéndolas con diversos enzimas y a continuación se electrodepositan selectivamente sobre la superficie de un electrodo. Como parte de esta metodología, se prepararon nanopartículas de oro biofuncionalizadas utilizando tres estrategias diferentes: a través del enlace dativo oro-tiol, por adsorción directa o mediante interacción electrostática siguiendo la técnica layer-by-layer (capa por capa). Para la funcionalización de las nanopartículas de oro se emplearon distintas biomoléculas, como los enzimas peroxidasa de rábano (HRP), glucosa oxidasa (GOX) y albúmina de suero bovino (BSA), y finalmente oligonucleótidos modificados con moléculas fluorescentes y grupos tiol. Las nanopartículas biofuncionalizadas fueron caracterizadas mediante técnicas de espectroscopía UV-visible, microscopía electrónica de transmisión (TEM) y medida del potencial zeta. Mediante espectroscopía UV-visible se observó un pico de resonancia de plasmón característico de las nanopartículas modificadas, relacionado con la estabilidad de la preparación. La medida del potencial zeta permitió la caracterización de las nanopartículas de oro modificadas capa por capa con polímero redox y enzimas. También se estudiaron los cambios en el potencial zeta de nanopartículas modificadas con BSA a distintos valores de pH. Tras la preparación de las partículas biofuncionalizadas, se llevaron a cabo estudios fundamentales de electrodeposición de nanopartículas de oro modificadas con BSA y un polímero redox, con el fin de analizar el efecto de varios parámetros: potencial aplicado, tiempo de deposición, distancia entre los electrodos, superficie del electrodo auxiliar y pH del medio. Para estudiar el comportamiento electrocatalítico de las nanopartículas modificadas una vez electrodepositadas, se llevaron a cabo experimentos utilizando coloides de oro modificados con HRP y GOX. A continuación se empleó esta metodología para el desarrollo de biochips, utilizando dos configuraciones diferentes. En la primera, se electrodepositaron nanopartículas de oro funcionalizadas con GOX y HRP y modificadas con un polímero redox sobre la superficie de un chip de electrodos interdigitados (IDE), consiguiendo eliminar por completo las repuestas no específicas. En la segunda configuración, las partículas se modificaron con una capa adicional de polímero redox, comprobando de nuevo la ausencia total de respuestas no específicas después de la electrodeposición. Esta método de patterning es genérico y puede utilizarse para la producción de diversos biochips. El segundo método de patterning también está basado en el control electroquímico, y consiste en la modificación de los electrodos con monocapas autoensambladas electroactivas cuya funcionalidad es modulable en función del potencial aplicado. En esta metodología, la monocapa electroactiva contiene grupos acetal que pueden ser desprotegidos selectivamente mediante la aplicación de un potencial en zonas específicas de la superficie del electrodo. De esta manera quedan expuestos en la superficie grupos aldehído activos, que pueden ser fácilmente conjugados con aminas primarias presentes en las biomoléculas de interés. Los enzimas GOX y HRP se usaron como proteínas modelo para comprobar la versatilidad de esta técnica. Su aplicabilidad para la fabricación de biochips se demostró con medidas amperométricas y medidas en tiempo real mediante resonancia de plasmón de superficie combinado con electroquímica (eSPR). La tercera metodología es también un sistema de patterning controlado electroquímicamente, pero en este caso se utiliza la inmovilización del 4,4-bipiridil como base para la creación de biochips. Se sintetizaron moléculas de 4,4-bipiridil funcionalizadas con grupos carboxílicos, que fueron caracterizadas electroquímicamente y a continuación conjugadas con las biomoléculas de interés para la creación de biochips. La selectividad de estos sistemas se demostró colorimétricamente, obteniéndose niveles mínimos de respuesta inespecífica. Por último, el cuarto de los métodos de patterning desarrollados está basado en la técnica de fotolitografía. Los enzimas glucosa oxidasa y sarcosina oxidasa se depositaron selectivamente junto con un polímero redox sobre la superficie de electrodos interdigitados utilizando un proceso de lift off, consiguiendo eliminar por completo las señales cruzadas o cross-talk. Como parte de esta metodología se optimizaron varios procedimientos de inmovilización de las biomoléculas, con el fin de seleccionar la estrategia más adecuada. También se llevaron a cabo ensayos con diferentes reactivos para eliminar la adsorción inespecífica. Finalmente, el sistema optimizado fue aplicado sobre IDEs fabricados mediante fotolitografía. Los sensores de glucosa y sarcosina respondieron de forma selectiva a sus respectivos sustratos, con ausencia total de cross-talk. La presente tesis está estructurada en 7 capítulos. En el Capítulo I se exponen las bases del desarrollo de biochips, métodos de patterning con control electroquímico, otros métodos de patterning selectivo y las técnicas de fotolitografía, así como un resumen de la tesis. El Capítulo 2 y 3 describe la síntesis de coloides de oro, la modificación con biomoléculas, los estudios de estabilidad y los estudios fundamentales de electrodeposición de las nanopartículas de oro modificadas sobre la superficie de los electrodos. En el Capítulo 4 se muestra la aplicación de la electrodeposición de nanopartículas de oro biofuncionalizadas para la creación de biochips. El Capítulo 5 describe la inmovilización selectiva de biomoléculas mediante la desprotección electroquímica de monocapas autoensambladas electroactivas. En el Capítulo 6 se muestra la síntesis, caracterización e inmovilización selectiva de derivados de 4,4- bipiridil funcionalizados con HRP. El Capítulo 7 describe el patterning selectivo en la escala micrométrica de dos oxidasas sobre un chip de electrodos interdigitados mediante fotolitografía. Finalmente, el Capítulo 8 resume las conclusiones y el trabajo futuro.
There is an increasing demand of multianalyte sensing devices having potential applications in biomedical, biotechnological, industrial and environmental fields. A good spatial control during biomolecule deposition step is strictly necessary; each biomolecule has to be precisely deposited on the surface of the relevant sensor (eg., an amperometric transducer), avoiding mixing that can compromise the biosensor specificity. The aim of this thesis is to develop different patterning methods for the selective immobilization of biomolecules. The first method is selective electrodeposition of biofunctionalized Au nanoparticles for biosensor arrays. This is an electrochemically controlled patterning method where the Au nanoparticles modified by the enzymes initially and later the enzyme modified Au nanoparticles were electrodeposited selectively on the electrode surface. As a part of this methodology, initially biofunctionalized Au nanoparticles were prepared using three different approcahes. One is Au-thiol dative bonding, the second is direct adsorption and finally electrostatic layerby- layer approach. Different biomolecules like horse radish peroxidase(HRP), glucose oxidase (GOX), bovine serum albumin(BSA), and finally fluorescence labelled oilgonucleotide thiols were used to attch to the Au nanoparticles. Biofunctionalized Au nanoparticles were characterized by different techniques like zeta sizer, UV-Vis spectroscopy, transmission electron microscopy (TEM). UV-Vis spectroscopy showed the successfull modification of Au nanoparticles with a characterstic surface plasmon peak related to the stability. By using zeta sizer, layer-by-layer modification of the Au nanoparticles with redox polymer and enzymes were characterized successfully. Changes of the Au nanoparticles modified with BSA was characterised at different pH s by using the zeta sizer. After the preparation of biofunctionalized particles, some fundamental studies were done with electrodeposition of Au nanoparticles modified with medically important BSA, redox polymer to see how different parameters like potential, time of deposition, interelectrode distance, counter electrode sized, pH, effect the electrodeposition. As a part of these fundamental studies Au colloids modified with HRP and GOX were deposited for studying the electrocalaytic behaviour of the enzymes on the Au nanoparticles after electrodeposition. Later this methodology was applied for creating biosensor arrays by using two different approaches. In the first approach, GOX and HRP functionalized redox polymer modified Au nanoparticles were electrodeposited successfully on an interdigitated electrode (IDE) array with complete absence of non-specific response. In the second approach the particles were modified with an extra redox polymer layer and proved that there is complete absence of nonspecific response after electrodeposition. Moreover, this patterning methodology is generic and can be used for production of different biochips. The second method is another electrochemically controlled patterning method where the electrodes were immobilized with self assembled monolayers with electroactive functionalities which can be tunable with potentials. In this methodology, electroactive self-assembled monolayer contains an active ligand aldehyde which can be readily conjugated to the primary amine group of the biomolecule is protected in the form of acetal. Later when a active potential was applied to the underlying electrode surface, the acetal functionality is deprotected to reveal the aldehyde functionality which was further conjugated to the biomolecule. Two enzymes GOX, HRP were used as model proteins to prove the versatility of this technique. Amperometric as well as real time measurements proved the selective applicability of this technique for creation of biosensor arrays. The third methodology is also an electrochemically controlled patterning methodology where the special advantage of the electrochemically-controlled immobilization of the 4,4-bipyridyl was taken as base for the creation of biosensor arrays. In this methodology, carboxylic acid functionalised 4,4, bipyridyl molecules were synthesized and characterized by electrochemistry. Later the biomolecules were conjugated to these special molecules for the creation of sensor arrays. Proof of selectivity was shown using colourimetrically with minimal non-specific response. Finally in the fourth method which is based on the photolithography technique, two different oxidases GOX & SOX were patterned along with redox polymer selectively on an IDE array using the lift off process with complete absence of cross-talk. As a part of this methodology, different immobilization methods were optimized initially for checking the best optimisation strategy. Later different reagents were tried to optimise the best reagent that prevents the non-specific adsorption. Later this optimised system was applied on the pholithographically created IDE array. Sarcosine and glucose sensors responded selectively to their substrates with complete absence of cross talk. This thesis is structured in 7 chapters. Chapter 1 establishes to basics of the biosensor arrays, electrochemically controlled patterning methods, other selectively patterned methods, photolithography and summary of this thesis. Chapter 2 describes about the gold colloid synthesis, modification with the biomolecules, stability studies. Chapter 3 decribes fundamental studies of the electrodeposition of the functionalised Au nanoparticles on the electrode surface. Chapter 4 describes the application of the electrodeposition of the protein functionalised Au nanoparticles for the creation of biosensor arrays. Chapter 5 describes the selective immobilization of biomolecules through electrochemical deprotection of electroactive self-assembled monolayers. Chapter 6 describes the synthesis, characterization and selective immobilization of HRP functionalized 4,4-bipyridyl derivatives. Chapter 7 describes the selective microscale protein patterning of two oxidases on an IDE array through photolithography. Finally chapter 8 summarizes the conclusions and the future work.

Choline is indispensable for a number of fundamental processes in the body. Besides being the precursor of the acetylcholine, an important neurotransmitter, choline is found in the cell membrane structure combining with fatty acids, phosphate and glycerol. Its deficiency may result in nervous system disorders, fatty acid build up in the liver, along with increased cholesterol levels, high blood pressure and memory loss. Thus, rapid detection methods are required for the determination of choline in biological fluids. In this study a choline oxidase biosensor was constructed for the determination of choline. During construction of the biosensor, glucose oxidase was used as a model enzyme, before choline oxidase used. The Teflon (PTFE) membrane of the oxygen electrode was grafted with 2-hydroxyethyl methacrylate (HEMA, 15%, v/v) in the presence of ferrous ammonium sulphate (FAS, 0.1%, w/v) by gamma irradiation and ethyleneglycol dimethacrylate (EGDMA, 0.15 %, v/v) was used as a crosslinker in a series of membranes. HEMA-grafted membranes were activated with epichlorohydrin or glutaraldehyde to maintain covalent immobilization of enzyme. The enzyme activity was measured with an oxygen electrode unit based on oxygen consumption upon substrate addition. Membranes were characterized in terms of grafting conditions and mechanical properties. Membranes, gamma irradiated in a solution of HEMA (15%) and FAS (0.1%) for 24 h, were found to be suitable for use in the further studies. Mechanical test results revealed that HEMA grafting made Teflon membrane more flexible and the presence of EGDMA made the grafted membrane stiffer. During optimization stage, it was found that the immobilized enzyme amount was not sufficient to obtain enzyme activity. Thus, the membrane preparation stage was modified to obtain thinner membranes. The immobilized glucose oxidase and choline oxidase contents on thin HEMA grafted membranes were determined by Bradford and Lowry methods. The influence of EGDMA presence and the epichlorohydrin activation duration on enzyme activity studies revealed that the membrane should be prepared in the absence of EGDMA and 30 min activation duration is appropriate for epichlorohydrin coupling. The study on the influence of membrane activation procedures revealed that the membranes activated with glutaraldehyde had a higher specific activity than the membranes activated with epichlorohydrin. Upon stretching membrane on the electrode directly rather than placing in the sample unit, the response of the enzyme immobilized sensor improved with high specific activity. The optimum choline oxidase concentration was found to be 2 mg/mL considering the effect of immobilization concentration on enzyme activity. With the choline oxidase biosensor, the linear working range was determined as 0.052-0.348 mM, with a 40 ±
5 µ
M minimum detection limit. The response of the sensor decreased linearly upon successive measurements.

Phenolic compounds are the chemicals which are used by many different industries and as a result of this spread to the environment. These compounds can be absorbed easily through the human and animal skin and through the mucosal membrane, mix in to the blood circulation and thus create a toxic effect on several tissue and organs including, liver, lung and kidneys. For this reason, determination of phenolic compounds emitted to environment is a very important issue. In fact, there are standard methods for the determination of these compounds like HPLC, Spectrophotometric and calorimetric methods however, these are time consuming methods and requires to be expertise. On the other hand, there are also different types of biosensors developed for the phenolic compound detection. In this study, a new, disposable, cheap and convenient tyrosinase biosensor was developed for the phenolic compound detection. By means of absorption method, the enzyme tyrosinase and the chromophore MBTH were immobilized on the support material and as a model substrate L- dopa was used. As a result of optimization studies 1mg/ml tyrosinase concentration and 1.5mM MBTH concentration were determined for using in biosensor construction. Detection limit of l-dopa, model substrate, found as 0,064 mM and for other phenolic compounds, 4-chlorophenol, catechol, m-cresol and p-cresol, detection limit was obtained 0.032 mM, 0.032 mM, 0.128 mM, 0.128 mM, respectively. In addition, we found that the biosensor response was not affected by pH changes ranging from 3 to 11. The stability of biosensor which is one of the important parameter for commercialization was not change through 70 days at room temperature and 4°
C when compared to at the beginning response.

With the era of technological change at its all time high, advancements in the field of photonics offer us a wide range of innovative potential applications. Over the years photonics has played an important role in modern industries such as telecommunication, sensors and medical imaging. One of the fields which has received a lot of attention is Photonic Crystal Fibers (PCF) for biosensor application. Photonic crystal fiber is a unique type of optical fiber in which continuous channels of (typically) air run their entire length. These 'holes' serve to both confine electromagnetic waves within the core of the fiber and to tailor its transmission properties. The classification of photonic crystal fiber can be solid core PCF where the light is guided by total internal reflection, and Hollow core photonic bandgap fibers (PBF) in which light is guided through the photonic bandgap effect. Simulation of PCF has been done through commercial software known as COMSOL which follows the finite element approach. The focus of this thesis is on the application of PCF for different sensing applications. Traditionally, solid core PCF has been used for sensing purposes as the cladding channels can be filled with gas or liquid, thus serving as an efficient type of evanescent wave sensing. Hollow core PBF offers huge improvements as the interaction between light and matter is increased by the presence of sample in the core where most of the light is confined. Conventionally, HC-PBF is used for sensor purposes by selectively filling the core. Here we have used a non-selective filling technique wherein all the channels of PCF are being filled with samples. When the fiber is empty it guides a particular band, and upon filling with other samples, the bandgap is shifted, and depending on this shift one can determine the refractive index of the sample. This type of sensor has been able to detect as low as 10 -5 change in refractive index just by taking a few centimeters of HC-PBF. Laser Flash Photolysis is one of the leading methods used in photochemistry to determine the transient species such as radicals, excited states or ions, in chemical and biological systems. By using HC-PBF we have replaced the conventional technique of LFP where in a test-tube is used to hold the sample. The sample is excited through a laser and a monitoring beam is used to observe the amount of absorption. The sample required here is on the order of a milliliter which can be scaled down to pico liter by the use of PCF. The LFP results using PCF showed signal enhancement of at least an order of magnitude for samples like xanthone in toluene, xanthone in acetonitryl and water soluble benzoin in methyl viologen. Raman Spectroscopy is yet another area which had a surge of growth for label free detection of samples. One of the reasons for its popularity is that it provides a unique optical fingerprint of chemicals and biomolecules. In this thesis we have focused on developing HC-PBF for enhancing the Raman signal from the sample. We have obtained an enhancement of over 40 times when using a HC-PBF with a length of 9.5cm. We have also used HC-PBF to study the enhancement of Raman signal from colloidal nanoparticles in an aqueous solution. Supercontinuum generation is yet another area which has seen tremendous growth through the use of solid core PCF. Here we have covered the excitation of cladding and core mode in an endlessly single mode PCF which has the potential to be used as an effective type of biosensor as the penetration of light in the cladding channels is very strong compared to an evanescent wave field.

The research described in this thesis concerns the investigation of technologies for the molecular engineering of the biosensor interface. Two avenues of investigation have been explored: the use of polymer matrices to modify the properties and functions of optical biosensor interfaces and the conjugation of photochromic dyes to protein systems to achieve photomodulation of protein function for biosensor applications. A comparison of the industry standard polymer, carboxymethyl dextran (CMD) was made against carboxymethyl cellulose and mixed systems, including a novel synthetic polymer, carboxylated polynoxylin. While CMD was found to provide the highest surface loading of protein, mixed polymers demonstrated the ability to allow prediction of surface loading, and showed features such as improved resistance to biological degradation. A novel method of depositing interfaces was investigated, allied to a study of liquid handling methods. A system was developed that allowed a printed heterogeneous array to be produced which showed preferential binding of specific analytes to defined areas of the sensor, whilst the other printed arrays retaining a high degree of non-specific interaction. The use of photochromic dyes to modulate protein function was applied to glucose oxidase and horseradish peroxidase. From the initial results, a hypothesis regarding the mechanism of photomodulation and its effect concerning the molecular weight of the conjugated protein was proposed. This was examined by the photomodulation of members of the peroxidase super family, antibodies and Fab fragments. From these results, the hypothesis was proved to be correct but incomplete, and was modified to include the disruption of the hydration shell around the protein caused by photochromic switching. Further research directly related to these experiments, and in novel fields of investigation have been proposed.

This project investigates the interaction of water with graphene and graphane. It also examines how the functionalization with polystyrene will affect the surfaces and their interaction with water. This study is completely theoretical and is performed with the simulating and modeling software called Materials Studio from Accelrys, Inc. Calculations are done with CASTEP, Dmol3 and Forcite. It is important to gain a deep understanding about the interactions between graphene (or graphane) surfaces and the water that is attached to it, since these materials are of a large interest for biosensor applications. The conclusion is that graphane (compared to graphene) is more susceptible to functionalization with styrene. This is most probably due to the sp3 hybridization of the C atoms, so one has to hydrogenate the graphene surface to make it more functionalizable. The graphane surface are also more hydrophilic than graphene. However, by functionalization with styrene it is possible to make both surfaces more hydrophilic and thus more suitable for biological system. Further investigations need to be done in order to validate these results.

Surface plasmon resonance (SPR) biosensors have attracted great attention in scientific research in the past three decades. Extensive studies on the immobilisation of biorecognition elements have been conducted in pursuit of higher sensitivity, but trialled formats have focussed on a thin layer modification next to the plasmon film, which usually requires in situ derivatization. This thesis investigates an 'off-chip' immobilisation strategy for SPR biosensing using silica particles and considers the implications of a particle-modified evanescent field on the signal amplitude and kinetics, for an exemplar affinity binding between immobilised IgG and its anti-IgG complement. Submicron silica particles were synthesized as carriers for the bio-recognition elements. They were then immobilised to form a sub-monolayer on the gold film of an SPR biosensor using two methods: thiolsilane coupling and physical adsorption aided by mechanical pressure. The bio-sensitivity towards an antigen/antibody interaction was lower than an SPR biosensor with an alkanethiolate SAM due to the difference in ligand capacity and position in the evanescent field. The binding kinetics of antigen/antibody pair was found to follow the Langmuir model closely in a continuous flow configuration but was heavily limited by the mass transport from the bulk to the sensor surface in a stop-flow configuration. A packed channel configuration was designed with larger gel particles as ligand carriers, packed on top of a gold film to create a column-modified SPR biosensor. This sensor has comparable bio-sensitivity to the previous sub-monolayer particle-modified systems, but the binding and dissociation of the analyte was heavily dependent on mass transport and binding equilibria across the column. A bi-directional diffusion mechanism was proposed based on a two-compartment mass transport model and the expanded model fitted well with the experimental data. The column-modified sensor was also studied by SPR imaging and analyte band formation was observed and analysed. Using the lateral resolution, a multiplexing particle column configuration was explored, and its potential in distinguishing a multicomponent analyte.

Thesis: M. Eng., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2015.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Title as it appears in MIT Commencement Exercises program, June 5, 2015: Portable impedimetric biosensor. Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 64-65).
Measuring proteins in blood is particularly common such as in PSA, to determine prostate function and in many other bio-applications. Today this process is inconveniencing in that it requires a lot of blood from the patients for tests to be made. Furthermore it is time consuming and expensive since samples are transferred to a central lab where tests are carried out on optical assays. Using electrical read out methods several measurements can be multiplexed and a differential measurements made. With a differential measurement the precision of the system is improved by cancelling out common mode bio-chemical noise. The multiplexed measurement enables measurement of several analytes. Using the embedded system MAX32600, an electrical impedance meter was designed that measures three impedances simultaneously with accuracy range of up-to 1% and precision of 0.2% of the actual impedance measured from an Impedance analyzer.
by Gerard Bamuturaki Kato.
M. Eng.

The ability to utilise new knowledge of biomarkers from genomic and proteomic data will have a great impact on molecular diagnosis. Biomarker detection could be achieved by utilising a capture molecule that associates specifically with the target biomarker. The work described in this thesis focuses on a platform comprising a lysozyme binding aptamer and an amperometric electrode (an electrochemical aptasensor). To couple the binding reaction to a change in current, the aptamer is modified with a redox group, ferrocene. Two types of signalling aptamer were constructed, one comprised the aptamer self-assembled on gold and hybridised to a short complementary oligonucleotide carrying a ferrocene group. The second incorporated the binding sequence into a molecular beacon, one end of which self-assembled onto the electrode, the other end carried the ferrocene group. Both of these showed a lysozyme dependent change in current on a gold electrode. Further characterisation of the first aptasensor suggested that the nucleic acid formed a multilayer structure on the electrode surface and that lysozyme binding induced conformational change moved ferrocene close to the surface, increasing the current. In contrast, the second aptamer usually showed a decrease in current in the presence of lysozyme suggesting that the binding resulted in the ferrocene moving away from the surface. In order to evaluate the possible use of these aptasensors for continuous in vivo measurement, needle shaped microelectrodes arrays were produced and the beacon aptamer immobilised on the surface. These electrodes had high impedance which resulted in low sensitivity, however lysozyme binding could still be detected using electrochemical impedance spectroscopy with ferrocyanide in solution. These microspike arrays could also be used for glucose sensing following modification with glucose oxidase.

Biosensors are nowadays a powerful tool to enable the detection of specific biological interactions and to evaluate the concentration dependence in the response. A biosensor usually consists of three different parts: the sample to be measured, the transducer and the electronic system that amplifies the signal, analyzes the data and brings a result to the final user. The transducer includes the bioreceptor (which specifically interacts with the sample) and the interface that transforms the recognition from the bioreceptor into a measurable signal. When the analyte interacts with the bioreceptor, the transducer sends a signal that is processed by the electronics. All this process occurs in an efficient, quick, cheap, easy, simple and specific way. Regarding the type of the transductor, the biosensors can be electrochemical, optical, acoustic, magnetic or thermometric; but overall the most powerful ones are the optical biosensors, and among them the grating coupler. As a technique for investigating processes at the solid/liquid interface, presents high mechanical stability, immunity to electromagnetic interferences and pushes the sensitivity to levels even higher than other techniques and allows for the direct monitoring of macromolecular adsorption. Taking advantage of the last advances in nanotechnology, the goal of this thesis is to study the versatility of an Optical Grating Coupler Biosensor. The design of new grating sensor chips will be investigated, a new calibration technique for the sensors will be proposed and, taking advantage of the technique, different biomedical scenarios will be tested.
Esta tesis consiste en el diseño, fabricación y test de un Biosensor Óptico basado en redes de difracción y sus aplicaciones en biomedicina. Los biosensores ópticos son dispositivos que detectan interacciones biomoleculares específicas mediante un transductor óptico. Exhiben alta sensibilidad, alta estabilidad mecánica, son inmunes a las interferencias electromagnéticas y permiten medidas no destructivas. En los Biosensores Ópticos por Onda Evanescente un modo guiado se propaga a lo largo de la guía de ondas mientras que la onda evanescente interactúa con la superficie del sensor, reconociendo cualquier interacción biomolecular que provoque una modificación en el índice de refracción efectivo de la guía óptica. En este caso, la inserción de luz láser en la guía óptica se produce con ayuda de una red de difracción grabada en la superficie del sensor. Para un ángulo muy preciso se excita un modo guiado. Como consecuencia de las reacciones en la superficie se produce un cambio en el ángulo de acoplo. La medida en tiempo real del ángulo de acoplo, en función de la actividad bioquímica en la superficie es la base de este tipo de biosensor óptico. El objetivo es fabricar sensores de bajo coste en polímero y también en distintos materiales que permitan calibrar otras técnicas. Otro objetivo de esta tesis es la calibración de los sensores y de las distintas soluciones buffer comúnmente usadas en biosensado. Como aplicación, se ha usado un equipo comercial (Optical Waveguide Lightomode Spectroscopy, OWLS, MicroVacuum) para estudiar, mediante control electroquímico, el crecimiento y la liberación de multicapas de PLL/DNA para aplicaciones en administración de fármacos. También se ha usado el OWLS para optimizar la inmovilización de receptores olfativos en un dispositivo biosensor para el desarrollo de una nariz bioelectrónica.

Thesis (M.S.)--West Virginia University, 2006.
Title from document title page. Document formatted into pages; contains vii, 85 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 70-72).

Biosensors are today important devices within various application areas. In this thesis a new type of label-free biosensor device is studied, which is fabricated using the same processes used for the fabrication of integrated circuits. This enables tighter integration and further sensors/biosensor miniaturization. The device is a so-called Thin Film Bulk Acoustic Resonator (FBAR). Within this thesis a low temperature reactive sputtering process for growing AlN thin films with a c-axis inclination of 20-30o has been developed. This enables shear mode FBAR fabrication suitable for in-liquid operation, essential for biosensor applications. Shear mode FBARs were fabricated operating at frequencies above 1GHz exhibiting Q values of 100-200 in water and electromechanical coupling factors kt2 of about 1.8%. This made it possible to move the thickness excited shear mode sensing of biological layers into a new sensing regime using substantially higher operation frequencies than the conventionally used quartz crystal microbalance (QCM) operating at 5-20MHz. Measured noise levels of shear mode FBARs in contact with water showed the resolution to be in the range 0.3ng/cm2 to 7.5ng/cm2. This demonstrated the FBAR resolution without any averaging or additional stabilization measures already to be in the same range as the conventional QCM (5ng/cm2), suggesting that FBARs may be a competitive and low cost alternative to QCM. The linear thickness limit for sensing of biomolecular layers was concluded to be larger than the thickness of the majority of the molecular systems envisaged for FBAR biosensor applications. A temperature compensated shear mode FBAR composite structure was demonstrated with retained coupling factor and Q-value by utilizing the second mode of operation. Understanding has been gained on the sensor operation as well as on how the design parameters influence its performance. Specifically, sensitivity amplification utilizing low acoustic impedance layers in the FBAR structure has been demonstrated and explained. Further, temperature compensated Lamb mode (FPAR) devices were also studied and demonstrated with optimized electromechanical couplings.
wisenet

Thesis (M.S.)--West Virginia University, 2007.
Title from document title page. Document formatted into pages; contains viii, 90 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 74-76).