Electrochemically controlled patterning for biosensor arrays.

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

PPG Heart Rate Detection in the Presence of Motion Artifacts

Peripheral circulation can elicit a lot of relevant diagnostic information like heart rate and blood oxygenation level without the need of any invasive measurements. Photoplethysmographic (PPG) signals are obtained by such non-invasive measurements using pulse oximeters. PPG signals, although non-invasive, come with some inherent problems. In a nonhospital environment, like when using a wearable type of sensor, a measured PPG signal predominantly suffers from motion artifacts. Ambient light conditions, temperature, and respiratory artifacts are a few other noise sources that affect the PPG signals when trying to measure heart rates. Most motion artifacts lie in the same frequency range as that of the required noise free signal. So, simple filtering is unlikely to work. This work explores adaptive filtering techniques that are commonly used for noise removal. The current work also proposes to use a popular active noise cancellation technique combined with adaptive filtering and artificial neural networks to minimize the motion artifacts. Furthermore, the work proposes a wrapper algorithm that covers the deficiency of the other techniques. Finally, this work employs a smart peak identification technique to measure reliable heart rates from the MA reduced signals

Formal models of the extension activity of DNA polymerase enzymes

The study of formal language operations inspired by enzymatic actions on DNA is part of ongoing efforts to provide a formal framework and rigorous treatment of DNA-based information and DNA-based computation. Other studies along these lines include theoretical explorations of splicing systems, insertion-deletion systems, substitution, hairpin extension, hairpin reduction, superposition, overlapping concatenation, conditional concatenation, contextual intra- and intermolecular recombinations, as well as template-guided recombination. First, a formal language operation is proposed and investigated, inspired by the naturally occurring phenomenon of DNA primer extension by a DNA-template-directed DNA polymerase enzyme. Given two DNA strings u and v, where the shorter string v (called the primer) is Watson-Crick complementary and can thus bind to a substring of the longer string u (called the template) the result of the primer extension is a DNA string that is complementary to a suffix of the template which starts at the binding position of the primer. The operation of DNA primer extension can be abstracted as a binary operation on two formal languages: a template language L1 and a primer language L2. This language operation is called L1-directed extension of L2 and the closure properties of various language classes, including the classes in the Chomsky hierarchy, are studied under directed extension. Furthermore, the question of finding necessary and sufficient conditions for a given language of target strings to be generated from a given template language when the primer language is unknown is answered. The canonic inverse of directed extension is used in order to obtain the optimal solution (the minimal primer language) to this question. The second research project investigates properties of the binary string and language operation overlap assembly as defined by Csuhaj-Varju, Petre and Vaszil as a formal model of the linear self-assembly of DNA strands: The overlap assembly of two strings, xy and yz, which share an overlap y, results in the string xyz. In this context, we investigate overlap assembly and its properties: closure properties of various language families under this operation, and related decision problems. A theoretical analysis of the possible use of iterated overlap assembly to generate combinatorial DNA libraries is also given. The third research project continues the exploration of the properties of the overlap assembly operation by investigating closure properties of various language classes under iterated overlap assembly, and the decidability of the completeness of a language. The problem of deciding whether a given string is terminal with respect to a language, and the problem of deciding if a given language can be generated by an overlap assembly operation of two other given languages are also investigated

PPG Heart Rate Detection in the Presence of Motion Artifacts

Peripheral circulation can elicit a lot of relevant diagnostic information like heart rate and blood oxygenation level without the need of any invasive measurements. Photoplethysmographic (PPG) signals are obtained by such non-invasive measurements using pulse oximeters. PPG signals, although non-invasive, come with some inherent problems. In a nonhospital environment, like when using a wearable type of sensor, a measured PPG signal predominantly suffers from motion artifacts. Ambient light conditions, temperature, and respiratory artifacts are a few other noise sources that affect the PPG signals when trying to measure heart rates. Most motion artifacts lie in the same frequency range as that of the required noise free signal. So, simple filtering is unlikely to work. This work explores adaptive filtering techniques that are commonly used for noise removal. The current work also proposes to use a popular active noise cancellation technique combined with adaptive filtering and artificial neural networks to minimize the motion artifacts. Furthermore, the work proposes a wrapper algorithm that covers the deficiency of the other techniques. Finally, this work employs a smart peak identification technique to measure reliable heart rates from the MA reduced signals

Stochastic Compositional Gradient Descent under Compositional constraints

This work studies constrained stochastic optimization problems where the objective and constraint functions are convex and expressed as compositions of stochastic functions. The problem arises in the context of fair classification, fair regression, and the design of queuing systems. Of particular interest is the large-scale setting where an oracle provides the stochastic gradients of the constituent functions, and the goal is to solve the problem with a minimal number of calls to the oracle. Owing to the compositional form, the stochastic gradients provided by the oracle do not yield unbiased estimates of the objective or constraint gradients. Instead, we construct approximate gradients by tracking the inner function evaluations, resulting in a quasi-gradient saddle point algorithm. We prove that the proposed algorithm is guaranteed to find the optimal and feasible solution almost surely. We further establish that the proposed algorithm requires $\mathcal{O}(1/\epsilon^4)$ data samples in order to obtain an $\epsilon$-approximate optimal point while also ensuring zero constraint violation. The result matches the sample complexity of the stochastic compositional gradient descent method for unconstrained problems and improves upon the best-known sample complexity results for the constrained settings. The efficacy of the proposed algorithm is tested on both fair classification and fair regression problems. The numerical results show that the proposed algorithm outperforms the state-of-the-art algorithms in terms of the convergence rate

Fabrication of High Surface Area Microporous ZnO from ZnO/Carbon Sacrificial Composite Monolith Template

Fabrication of porous materials from the standard sacrificial template method allows metal oxide nanostructures to be produced and have several applications in energy, filtration and constructing sensing devices. However, the low surface area of these nanostructures is a significant drawback for most applications. Here, we report the synthesis of ZnO/carbon composite monoliths in which carbon is used as a sacrificial template to produce zinc oxide (ZnO) porous nanostructures with a high specific surface area. The synthesized porous oxides of ZnO with a specific surface area of 78 m(2)/g are at least one order of magnitude higher than that of the ZnO nanotubes reported in the literature. The crucial point to achieving this remarkable result was the usage of a novel ZnO/carbon template where the carbon template was removed by simple heating in the air. As a high surface area porous nanostructured ZnO, these synthesized materials can be useful in various applications including catalysis, photocatalysis, separation, sensing, solar energy harvest and Zn-ion battery and as supercapacitors for energy storage

Enriched cell-free and cell-based native membrane derived vesicles (nMV) enabling rapid in-vitro electrophysiological analysis of the voltage-gated sodium channel 1.5

Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.5 (hNaV1.5; SCN5A) in 3 h. Subsequently, CB-nMVs were isolated from fractions of nitrogen-cavitated CHO cells overexpressing the hNaV1.5. In an integrative approach, nMVs were micro-transplanted into Xenopus laevis oocytes. CB-nMVs expressed native lidocaine-sensitive hNaV1.5 currents within 24 h; CF-nMVs did not elicit any response. Both the CB- and CF-nMV preparations evoked single-channel activity on the planar lipid bilayer while retaining sensitivity to lidocaine application. Our findings suggest a high usability of the quick-synthesis CF-nMVs and maintenance-free CB-nMVs as ready-to-use tools for in-vitro analysis of electrogenic membrane proteins and large, voltage-gated ion channels