Image segmentation is the process by which an image is divided into number of regions. The regions are to be homogeneous with respect to some property. Definition of homogeneity depends mainly on the expected patterns of the objects of interest. The algorithms designed to perform these tasks can be divided into two main families: Splitting Algorithms and Merging Algorithms. The latter comprises seeded region growing algorithms which provide the basis for our work. Seeded region growing methods such as Marker initiated Watershed segmentation depend principally on the quality and relevance of the initial seeds. In situations where the image contains a variety of aggregated objects of different shapes, finding reliable initial seeds can be a very complex task. This thesis describes a versatile approach for finding initial seeds on images featuring objects distinguishable by their structural and intensity profiles. This approach involves the use of hierarchical trees containing various information on the objects in the image. These trees can be searched for specific pattern to generate the initial seeds required to perform a reliable region growing process. Segmentation results are shown in this thesis. The above image segmentation scheme has been applied to detect isolated living cells in a sequence of frames and monitor their behavior through the time. The tissues utilized for these studies are isolated from the scales of Betta Splendens fish family. Since the isolated cells or chromatophores are sensitive to various kinds of toxic agents, a creation of cell-based toxin detector was suggested. Such sensor operation depends on an efficient segmentation of cell images and extraction of pertinent visual features. Our ultimate objective is to model and classify the observed cell behavior in order to detect and recognize biological or chemical agents affecting the cells. Some possible modelling and classification approaches are presented in this thesis. / Graduation date: 2003

To protect the environment and save human lives, the detection of various hazardous toxins of biological or chemical origin has been a major challenge to the researchers at Oregon State University. Living fish cells can indicate the presence of a wide range of toxins by reactions such as changing color and shape changes. A research team in Electrical and Computer Engineering Department is developing a hybrid detection device (Cytosensor) that combines biological reaction and digital technology. The functions of Cytosensor can be divided into three parts, which are real-time image acquisition, data processing and statistical data analysis. User-friendly Web-Based Distributed Applications (WBDA) for Cytosensor offer various utilities. WBDA allow the users to control and observe the local Cytosensor, search and retrieve data acquired by the sensor network, and process the acquired images remotely using only a web browser. Additionally, these applications minimize the user's exposure to dangerous chemicals or biological products. This thesis describes the design of a remote controller, system observer, remote processor, and search engine using JAVA applets, XML, Perl, MATLAB, and Peer-to-Peer models. Furthermore, the implementations of image segmentation technique in MATLAB and the Machine Vision Algorithm in JAVA for independent web-based processing are investigated. / Graduation date: 2003

Detection of biomolecules is important for the diagnosis and treatment of diseases. Low concentration detection, specific biomolecule detection, and point-of-care use are appealing characteristics for biosensors because of the possibility of early detection and quick results of specific biomolecules. Furthermore, inexpensive biosensors are appealing so that they are accessible to the general population. The biosensors in this study have the potential to satisfy these characteristics. In this study graphene field-effect transistors (G-FET) were fabricated. Graphene was grown using chemical vapor deposition (CVD) and transferred to a silicon/silicon oxide substrate. The CVD method is the most scalable and cost-effective method of producing graphene for devices. Standard photolithography was used to pattern and then deposit metal electrodes. Two separate experiments were conducted; one using electrostatic attraction to bind protein to the active area of the G-FET to detect the protein poly-L-lysine (PLL) and one using an aptamer modified G-FET to selectively detect the protein thrombin. Analyte was delivered using a homebuilt, pressure driven, microfluidic, mass flow system. Both experiments showed a detection of the protein. The PLL experiment showed a clear change in the effective gate voltage of the G-FET. The thrombin experiment showed a change in the effective gate voltage that varied with differing concentrations of thrombin present. Furthermore, in the thrombin experiment by changing from a thrombin solution back to buffer the effective gate voltage was brought back to its original value. A competing protein was introduced and gave a signal comparable to the signal of a 10 times smaller concentration of thrombin. All of this shows that CVD grown graphene in a FET biosensor can be used for protein detection. Furthermore, the specific detection of thrombin suggests that aptamer modified G-FETs with CVD grown graphene can be used as a protein specific biosensor. / Graduation date: 2013

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 170-185).

Durant la tesi doctoral s'ha comprovat que a partir de biosensors fabricats mitjançant una tecnología relativament senzilla i económica, com és la formació de matrius i la impressió serigràfica de tintes, es poden realitzar determinacions de diversos analits d'interès en l'àmbit alimentari no tan sols en solucions estàndard sinó també en mostres reals. Els resultats aconseguits suposen una important millora en l'abaratiment dels costos d'anàlisi i la reducció del temps necessari.El treball realitzar s'ha portat a terme en dues parts molt diferenciades; en una primera s'han desenvolupat biosensors screen-printing basats en resines epoxi per a l'anàlisi de pesticides i en una segona biosensors screen-printing basats en matrius de sílice (sol-gel) per a la determinació d'àcid màlic i làctic en vins.

La present tesi doctoral recull l'estudi de diverses modificacions químiques de sensors amperomètrics, adreçades cap a cercar solucions per als problemes típicament implícits en el desenvolupament de biosensors basats en enzims oxidoreductases, alhora que es busca la compatibilitat d'aquestes modificacions amb les estratègies d'immobilització enzimàtica emprades.La primera part del treball inclou el desenvolupament de quimiosensors reproduïbles i estables per a la determinació del cofactor NADH, del qual depenen els enzims deshidrogenases, i del compost peròxid d'hidrogen, obtingut com a producte de les reaccions en què participen gran part dels enzims oxidases. Els quimiosensors per a la determinació de NADH s'han basat en l'ús de diferents mediadors d'oxidació-reducció, mitjançant vàries estratègies d'incorporació als sistemes de detecció amperomètrica (en solució, per adsorció sobre la superfície electròdica o en membranes de Nafió, incorporats en matrius polimèriques de compòsits de grafit-epoxi, electropolimeritzats o immobilitzats en membranes de polisulfona). Els quimiosensors basats en membranes de polisulfona han mostrat nombrosos avantatges respecte la resta de quimiosensors desenvolupats. De fet, la polisulfona es presenta en aquest estudi com a un bon material polimèric per al desenvolupament de quimiosensors amperomètrics, atès que aconsegueix evitar els problemes de passivació de la superfície electròdica implícits en la determinació del cofactor NADH, i al mateix temps permet una retenció excel·lent dels mediadors immobilitzats al seu interior, amb una absència total de pèrdues d'aquestes espècies per dissolució. D'altra banda, en relació al desenvolupament de quimiosensors per a la determinació de peròxid d'hidrogen, s'han sintetitzat gels de sílice que incorporen diferents metalls que actuen com a catalitzadors dels processos d'oxidació i reducció del peròxid d'hidrogen. Aquests gels s'han dipositat com a mescles del corresponent xerogel amb acetat de cel·lulosa i polietilenglicol, a fi d'aconseguir membranes que minimitzen les limitacions d'aquest tipus de materials (formació d'esquerdes, absorció d'aigua,...). S'han emprat diferents tècniques d'anàlisi amb l'objectiu de dur a terme estudis de caracterització dels quimiosensors basats en membranes de polisulfona i en xerogels modificats amb metalls.La segona part del treball es va dedicar al desenvolupament de biosensors basats en diversos enzims oxidoreductases, mitjançant l'ús dels quimiosensors prèviament desenvolupats. Els quimiosensors per a NADH s'han adaptat per al desenvolupament de biosensors per a lactat, basats en la incorporació de l'enzim L-lactat deshidrogenasa en matrius de gels de sílice i en membranes de polisulfona, i de biosensors per a ió amoni, basats en la incorporació de l'enzim glutamat deshidrogenasa en polímers de mediador o en membranes de polisulfona. També es va desenvolupar un biosensor bienzimàtic per a la determinació d'urea, basat en la incorporació dels enzims glutamat deshidrogenasa i ureasa en membranes de polisulfona. D'altra banda, els quimiosensors per a peròxid d'hidrogen s'han emprat per al desenvolupament de biosensors per a glucosa, basats en la incorporació de l'enzim glucosa oxidasa en gels de sílice mitjançant vàries configuracions diferents. Els biosensors desenvolupats han demostrat la capacitat de les membranes de polisulfona i dels gels de sílice per a incorporar enzims. A més, en alguns casos, com el biosensor per a ió amoni, s'han aconseguit unes característiques analítiques excel·lents (sensibilitat elevada, intervals lineals amplis, temps de resposta curts, bona reproductibilitat entre corbes de calibració successives,...).Finalment, la darrera part del treball es va basar en l'adaptació dels quimiosensors i biosensors desenvolupats, basats en una configuració cilíndrica, a una configuració plana, mitjançant processos de serigrafia, i la seva posterior implementació en sistemes de flux. / The aim of this work was the study of different chemical modifications of amperometric sensors in order to minimise the problems involved in the development of biosensors based on oxidoreductase enzymes, trying to find compatibility between the used chemical modifications and the employed enzymatic immobilisation strategies.The first part of the work was devoted to the development of reliable and stable chemosensors for the determination of NADH cofactor and hydrogen peroxide, for the further development of dehydrogenase- and oxidase-based biosensors, respectively, since dehydrogenase enzymes are NAD-dependent and most of the oxidase enzymes involve hydrogen peroxide as a reaction product. Chemosensors for the determination of NADH were based on the incorporation of different electron mediators, using several incorporation strategies into the amperometrical detection system (in solution, by adsorption onto the electrode surface or onto Nafion membranes, by incorporation inside polymeric matrices of graphite-epoxy composites, by electropolymerization or by immobilisation inside polysulfone membranes). Chemosensors based on polysulfone membranes have shown many advantages in front of the other developed chemosensors. In fact, polysulfone is presented for the first time as an adequate polymeric material for the development of amperometric chemosensors, since it avoids the fouling surface problems typically involved in the amperometric determination of NADH cofactor, while at the same time allows an excellent retention of the immobilised mediators inside the membrane, without leakage of the immobilised species into the solution. On the other hand, in relation to the development of chemosensors for the determination of hydrogen peroxide, several sol-gels have been synthesised, which incorporate different metals acting as catalysts for the oxidation and reduction processes of hydrogen peroxide. These sol-gels have been mixed with cellulose acetate and polyethyleneglycol in order to be deposited as membranes, minimising the limitations of this kind of materials (cracking, water absorption,...). Different analysis techniques have been used with the aim of characterising the final chemosensors based on polysulfone membranes and metal-modified-xerogels. The second part of the work was directed towards the development of biosensors based on different oxidoreductase enzymes, using the previously developed chemosensors. Chemosensors for NADH have been used for developing lactate biosensors, based on the incorporation of L-lactate dehydrogenase enzyme inside sol-gel matrices and polysulfone membranes, and ammonium biosensors, based on the entrapment of glutamate dehydrogenase enzyme inside electropolymerized mediators or polysulfone membranes. Furthermore, a urea bienzymatic biosensor was developed, based on the incorporation of glutamate dehydrogenase and urease enzymes inside polysulfone membranes. On the other hand, chemosensors for hydrogen peroxide have been used for developing glucose biosensors, based on the immobilisation of glucose oxidase enzyme in sol-gels using different configurations. Both strategies, based on polysulfone membranes and sol-gels, have shown the ability of these membranes to incorporate enzymes into the biosensor configuration. Additionally, some of the developed biosensors, such as the ammonium biosensor, have achieved excellent analytical characteristics (high sensitivity, wide linear ranges, fast response times, good reproducibility among successive calibration curves,...).Finally, the last part of this work was based on the application of screen-printing technology for the preparation of the developed chemosensors and biosensors with a planar configuration, and their further implementation in flow systems.

En una primera parte de esta tesis fue desarrollado un nuevo electrodo a base de pasta de grafito-epoxi composite (GECE) conteniendo nitrato de bismuto [Bi(NO3)3] como precursor de bismuto incorporado [Bi(NO3)3-GECE)], como una posible alternativa para el análisis electroquímico por redisolución de metales pesados en cantidades traza. Los resultados claramente muestran las ventajas del Bi(NO3)3-GECE en combinación con la técnica de voltamperometría de redisolución anódica de onda cuadrada (SWASV) para la detección de metales pesados. Se llevaron a cabo medidas individuales y simultáneas de Pb y Cd y los resultados mostraron claramente las ventajas del Bi(NO3)3-GECE en combinación con la técnica SWASV para la detección de metales pesados. Con el uso del Bi(NO3)3-GECE construido se pueden realizar análisis rápidos y eficaces de iones de metal en cantidades traza como Pb y Cd entre otros en muestras ambientales de suelo, aguas naturales y aguas residuales. La ventaja inherente de la no necesidad de mercurio elimina muchas de las objeciones para el uso de métodos electroquímicos en la detección de tales especies en estos medios. Comparando el Bi(NO3)3-GECE con el electrodo de película de mercurio comúnmente usado y electrodo de película de bismuto desarrollado antes por nuestro grupo, el nuevo electrodo propuesto ofrece un notable funcionamiento en el análisis de metales pesados en cantidades traza, que puede ser de gran ventaja en electroquímica, contribuyendo a una aplicabilidad más amplia de técnicas electroquímicas por redisolución relacionadas con electrodos "sin mercurio". Además de aplicaciones ambientales el electrodo desarrollado basado en bismuto tendría interés especial para la aplicación en la detección de puntos cuánticos (QDs) basados en metales pesados. Tales aplicaciones están actualmente en proceso de estudio en nuestro grupo de investigación para la detección de ADN.Las otras partes de la tesis se dedican al desarrollo de nuevos sensores de ADN y proteínas basados en la misma técnica electroquímica de redisolución y el uso de nanopartículas de oro como marcas. Actualmente la detección electroquímica de secuencias de ADN específicas vía el evento de hibridación es una cuestión importante por lo cual diversas estrategias han sido propuestas.Genosensores electroquímicos de afinidad basados en el marcaje con nanopartículas de oro (AuNPs) y el uso de partículas paramagnéticas (MB) como plataforma para la inmovilización de la sonda de ADN de captura también han sido desarrollados en esta tesis a fin de demostrar la inducción magnética eficaz de un nuevo electrodo de grafito-epoxi composite-magnético (M-GECE) el cual fue construido también con pasta de grafito-epoxi composite con un pequeño imán de neodimio integrado.Todos los ensayos para la detección electroquímica de la hibridación del ADN desarrollados en esta tesis fueron basados en la detección directa de las marcas de AuNPs por medio de la técnica de voltametría de pulso diferencial (DPV) usando el M-GECE donde la intensidad de la corriente de la señal generada es directamente proporcional a la cantidad de ADN en la muestra. Como también ha sido demostrado, con el sensor de ADN asistido magnéticamente, el ADN analito condujo a una muy bien definida señal mientras que esencialmente ninguna señal fue observada para el ADN no complementario.Un nuevo inmunoensayo electroquímico sensible ha sido desarrollado, también basado en AuNPs como marca y MB como plataforma. El método fue evaluado para un inmunoensayo heterogéneo no competitivo de una IgG humana como proteína modelo. La detección electroquímica fue llevada a cabo en la misma forma que lo fue para ADN.La detección electroquímica de marcas de AuNPs en biosensores de afinidad usando métodos de redisolución permite el estudio detallado de la hibridación de ADN así como también inmuno-reacciones con interés en aplicaciones relacionadas con genosensores o inmunosensores. Los métodos electroquímicos usados para la detección de AuNPs como marca pueden ser muy prometedores tomando en cuenta su sensibilidad alta, límite de detección bajo, selectividad, simplicidad, bajo coste, y disponibilidad de instrumentos portátiles.Como conclusión final, las estrategias de análisis electroquímico de ADN y proteínas fueron demostradas con éxito y debido a los resultados prometedores su uso en muestras reales es viable. Tales biosensores de ADN e inmunosensores dan lugar a un enorme potencial de aplicación principalmente para diagnóstico clínico y monitoreo ambiental entre otros campos. / In the first part of this thesis a new graphite-epoxy composite electrode containing bismuth nitrate [Bi(NO3)3-GECE)], as built-in bismuth precursor as a possible alternative for electrochemical stripping analysis of trace heavy metals has been developed. Individual and simultaneous measurements of Pb and Cd were carried out and the results clearly showed the advantages of the Bi(NO3)3-GECE in combination with square wave anodic stripping voltammetry (SWASV) technique for heavy metals detection. Fast and effective analyses of trace metal ions such as Pb and Cd among others in environmental samples of soil, natural waters and effluents can be carried out by using the new Bi(NO3)3-GECE constructed. The inherent advantage of no necessity of mercury removes many of the objections for the use of the developed sensor. When comparing the Bi(NO3)3-GECE with the commonly used mercury film electrode and previously developed bismuth film electrode, the newly proposed electrode offers a remarkable performance in analysis of trace heavy metals, which can be advantageous in electrochemical, hence contributing to the wider applicability of electrochemical stripping techniques in connection with "mercury-free" electrodes. Beside environmental applications the developed bismuth based electrode would have special interest for application to heavy metal based quantum dots. Such applications are currently in the studying process at our research group for DNA detection.The other parts of the thesis are dedicated to the application of electrochemical stripping analysis in connection to gold nanoparticles for DNA and protein detection. Currently the electrochemical detection of specific DNA sequences via hybridization event is an important issue by which diverse strategies have been proposed. Affinity electrochemical genosensors based on labelling with gold nanoparticles (AuNPs) and the use of paramagnetic beads (MB) as platform for the immobilization of capture DNA probe have been also developed in this thesis in order to demonstrate the effective magnetic triggering of a new magnetic-graphite epoxy composite electrode (M-GECE) which was constructed with graphite-epoxy composite paste, with a small neodymium magnet integrated.All the assays for the DNA hybridization electrochemical detection developed in this thesis were based on the direct detection of AuNPs labels (anchored onto the M-GECE) by means of differential pulse voltammetry (DPV). The intensity of the generated current is directly proportional to the amount of DNA at the sample. As also has been demonstrated, with this magnetically assisted DNA sensor, target DNA leaded to very well defined signal whereas essentially no signal was observed for non-complementary DNA. By the other side a novel, sensitive electrochemical immunoassay has been also developed based in AuNPs as label and MB as platform. The method was studied and evaluated for a noncompetitive heterogeneous immunoassay of a human IgG as a model protein. The electrochemical detection was carried out in the same way that as for DNA.The electrochemical detection of AuNPs labels in affinity biosensors using stripping methods allows the detailed study of DNA hybridization as well as immunoreactions with interest in genosensor or immunosensor applications. The developed detection methodologies may be very promising taking into account their high sensitivity, low detection limit, selectivity, simplicity, low cost, and availability of portable instruments.As final conclusion, the DNA and protein electrochemical analysis strategies were successfully demonstrated and according to the promising results obtained its use for real samples is viable. Such DNA biosensors and immunosensors hold an enormous application potential principally for clinical diagnostic and environmental monitoring among other fields.

The assessment of the electrical properties of biomolecules at the nanoscale becomes necessary for gathering previous basic knowledge and for the control of the biosensor fabrication. I developed instrumentation, protocols, and theoretical frameworks for the nanoscale electrical characterization of biomolecules by AFM. Two novel types of AFM electrical characterizations were developed: electron transport through the biomolecules and dielectric polarization of the biomolecules (each one requires different instrumentation, protocols and theory). I succeeded in obtaining important electrical information on individual biomolecules with implications in electrical biosensor fabrication.KEY WORDS: AFM, Protein, Electrical, Biosensor

In this thesis, a novel electrochemical array is reported. The array consists of two planar halves, each having four carbon screen-printed band electrodes (SPEs), orthogonally facing each other and separated by a spacer to yield 16 two-electrode electrochemical cells with 1 mm² working electrode areas. The 16 counter electrodes were converted to Ag/AgCl by electrodeposition and anodization. These electrodes were stable for at least 30 days with potentials under the current densities used in our experiments. The 16 working electrodes were modified by Au electrodeposition, and were examined by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). <br /><br /> Immobilization strategies for biomolecules are of paramount importance for successful fabrication of biosensors. This thesis reports a new immobilization method that is based on patterned deposition of alkyl thiosulfates (Bunte salts). Monolayers were formed through electrochemical oxidation of Bunte salts at Au-modified electrodes. Single-component and mixed monolayers were investigated, where the mixed monolayers involved one component with a terminal carboxylic acid functional group to allow immobilization of biomolecules. <br /><br /> Applications of the newly developed immobilization method to an enzyme-based biosensor and an immunosensor were investigated. Glucose and biotin were chosen as model analytes, respectively. Glucose oxidase (GOx) and avidin were covalently immobilized onto the mixed-monolayer-modified electrodes through the carboxylic acid groups. Under the optimized conditions for the fabrication and operation of the biosensors, the new electrochemical array showed linearity up to 10 mM glucose with a sensitivity of 4. 7 nA mM^-1 and a detection limit of 0. 8 mM (S/N=3), and linearity up to 12. 8 µM biotin with a detection limit of 0. 08 µM (S/N=3).

In this thesis, a novel electrochemical array is reported. The array consists of two planar halves, each having four carbon screen-printed band electrodes (SPEs), orthogonally facing each other and separated by a spacer to yield 16 two-electrode electrochemical cells with 1 mm² working electrode areas. The 16 counter electrodes were converted to Ag/AgCl by electrodeposition and anodization. These electrodes were stable for at least 30 days with potentials under the current densities used in our experiments. The 16 working electrodes were modified by Au electrodeposition, and were examined by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). <br /><br /> Immobilization strategies for biomolecules are of paramount importance for successful fabrication of biosensors. This thesis reports a new immobilization method that is based on patterned deposition of alkyl thiosulfates (Bunte salts). Monolayers were formed through electrochemical oxidation of Bunte salts at Au-modified electrodes. Single-component and mixed monolayers were investigated, where the mixed monolayers involved one component with a terminal carboxylic acid functional group to allow immobilization of biomolecules. <br /><br /> Applications of the newly developed immobilization method to an enzyme-based biosensor and an immunosensor were investigated. Glucose and biotin were chosen as model analytes, respectively. Glucose oxidase (GOx) and avidin were covalently immobilized onto the mixed-monolayer-modified electrodes through the carboxylic acid groups. Under the optimized conditions for the fabrication and operation of the biosensors, the new electrochemical array showed linearity up to 10 mM glucose with a sensitivity of 4. 7 nA mM^-1 and a detection limit of 0. 8 mM (S/N=3), and linearity up to 12. 8 µM biotin with a detection limit of 0. 08 µM (S/N=3).