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Study on Oxidase/Peroxidase-based Biosensors with Pentacyanoferrate-bound Polymer / ペンタシアノ鉄錯体ポリマーを用いた酸化酵素/ペルオキシダーゼ型バイオセンサに関する研究Nieh, Chi-Hua 24 September 2013 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17895号 / 農博第2018号 / 新制||農||1017(附属図書館) / 学位論文||H25||N4791(農学部図書室) / 30715 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 加納 健司, 教授 三芳 秀人, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM
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Novel chemical labeling methods for analysis of protein function and cellular environment / 新規化学修飾法による蛋白質機能と細胞環境の解析Nishikawa, Yuki 25 March 2019 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21799号 / 工博第4616号 / 新制||工||1719(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 浜地 格, 教授 跡見 晴幸, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM
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Purification of Phage-Displayed HSA-Specific Peptide for Biosensor ProductionHuber, Alexander Domenico 05 June 2019 (has links)
No description available.
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Organic Electrochemical TransistorsKaphle, VIkash 17 December 2019 (has links)
No description available.
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Bioconjugation Techniques and Experimental Processing of Myeloperoxidase Detection SystemWang, Daniel 02 June 2020 (has links)
No description available.
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Determinación de carbohidratos en jugos de fruta con electrodos enzimáticosRistoff, María Emilia 22 November 2013 (has links)
El objetivo general de la presente tesis fue el diseño y la construcción de un biosensor amperométrico para determinar glucosa en jugos de fruta (utilizando el de manzana como modelo) que sea económico, de fácil construcción y que tenga respuesta rápida. Con tal fin se construyó un electrodo de pasta de carbono que consistió en un cilindro de teflón con un contacto eléctrico de acero inoxidable. Los materiales de la pasta de carbono fueron una mezcla de polvo fino de grafito y parafina, a los cuales se agregó la enzima glucosa oxidasa (GOx) y el ferroceno, como mediador. Se estudiaron diferentes condiciones de operación del biosensor tales como el pH del medio de reacción, el potencial aplicado y la carga de enzima en la pasta de carbono, siendo las condiciones óptimas seleccionadas: medio de reacción (buffer de fosfatos pH 7), potencial de trabajo (0.16 V) y carga enzimática (10%).
Se determinó la actividad enzimática de la enzima tanto libre como luego del proceso de inmovilización, y se estudió la cinética de la enzima en el electrodo, la cual se corresponde con la cinética típica de Michaelis-Menten, siendo los valores de los parámetros cinéticos k’m= 0.0195 M e imax= 35.5 μA.
A continuación se construyó la curva de calibración cuyo intervalo lineal de concentraciones de glucosa fue de 0.1 mM a 4.48 mM, con una sensibilidad de 0.61 μA/mM y un valor del límite de detección de 0.05 mM, valores completamente aceptables para el biosensor en cuestión. También se evaluaron características del sensor como su tiempo de respuesta, que fue menor a 12 segundos en todos los casos y la repetibilidad y la precisión intermedia dando en ambos casos muy buenos resultados.
Posteriormente se evaluó la estabilidad del sensor bajo diferentes condiciones de almacenamiento con y sin inmersión en la solución buffer y tanto bajo refrigeración como a temperatura ambiente. Todos los resultados fueron altamente satisfactorios y concuerdan con los esperados para el biosensor y más aún teniendo en cuenta la simpleza respecto de los materiales empleados y a la construcción del mismo.
Otro estudio fue la evaluación de las interferencias debidas a sustancias electroquímicamente activas en el jugo de fruta, en este caso de manzana, que puedan afectar a la señal de la glucosa. Como posibles interferentes se estudiaron: ácido ascórbico, sacarosa, fructosa, almidón, ácido málico y ácido cítrico. Sólo el ácido ascórbico fue una sustancia interferente, debiendo eliminarse este compuesto del jugo como paso previo a la determinación de glucosa con el biosensor. Se estudiaron dos técnicas de eliminación: eliminación enzimática del ácido ascórbico y eliminación por oxidación directa con oxígeno del aire ambiente. Ambas técnicas dieron excelentes resultados en la eliminación de este compuesto.
Finalmente se procedió a la determinación de glucosa en muestras reales, para lo cual se emplearon dos jugos de manzana: uno comercial y uno concentrado obtenido directamente de fábrica. Para validar los resultados obtenidos con el biosensor se utilizó un glucómetro digital. En todos los casos los errores en las determinaciones respecto a los valores obtenidos con el glucómetro fueron menores al 4%, lo que indica el muy buen desempeño del biosensor. / The main objective of the present Thesis was the design and construction of an amperometric biosensor to determine glucose in fruit juices (using apple juice as a model), considering this type of bio-electrodes are relatively cheap, simple to develop, set up and run and characterized by a fast response. The used carbon paste electrode consisted in a Teflon cylinder with stainless steel electric contact. Carbon paste was a mixture of graphite in fine powder and liquid paraffin, to which the enzyme glucose oxidase (GOx) and ferrocene as mediator was added. Several operation conditions were studied such as reaction medium pH, working potential and enzymatic load in the carbon paste, being the optimal conditions selected phosphate buffer (pH 7) as reaction medium, a working potential of 0.16 V, and 10% enzymatic load.
Enzymatic activity of both free enzyme and enzyme after the immobilization process was determined. Furthermore the kinetic of the enzyme in the electrode was studied, and the result shows that it follows a typical Michaelis-Menten kinetic, with k’m= 0.0195 M and imax= 35.5 μA.
The calibration curve shown that the linear range of glucose concentration was in the range 0.1 mM to 4.48 mM, with a sensitivity of 0.61 μA/mM and a detection limit of 0.05 mM. These values are completely acceptable for this kind of biosensor. Sensor characteristics, including response time (<12 sec. in all cases), intermediate precision and repeatability were evaluated.
Stability of the sensor under different storage conditions was also evaluated. Results were highly satisfactory and consistent, considering used materials and simplicity of construction. Interferences from electrochemically-active substances in apple juice, which may affect glucose signal, were carefully considered. The possible studied interfering substances were: ascorbic acid, sucrose, fructose, starch, malic acid and citric acid. Only the ascorbic acid was observed to be an interfering substance, which must be removed from juice previously to the determination of glucose with the biosensor. Two removal techniques for ascorbic acid were assayed: enzymatic removal and elimination by direct oxidation with air. Both techniques showed to be appropriate for ascorbic acid elimination.
Finally, the determination of glucose in real samples was carried out, using two apple juices samples, a ready to drink commercial one, and a concentrated juice directly obtained from a factory. To validate the results obtained with the biosensor a digital glucometer was used. In all cases the errors in the determinations were lower than 4%, demonstrating the very good performance of the biosensor.
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Developing signal enhancement strategies for photoelectrochemical nucleic acid sensingSaha, Sudip January 2021 (has links)
Recently, photoelectrochemical (PEC) signal transduction, with optical excitation and electronic readout, has been identified as a powerful transduction strategy for bioanalysis due to its high sensitivity and low limit-of-detection. Semiconductive materials have been used as the building blocks of PEC transducers, while plasmonic nanoparticles (NPs) are frequently used as signal amplifiers in these biosensors. Though these approaches have been previously used in PEC biosensing, the interaction between plasmonic and semiconductors NPs linked together through biomolecules are not currently well-understood. Herein, we developed new strategies for preparing photoelectrodes using solution-based methods to enhance the photocurrent of PEC transducers. These transducers were then used to investigate the interaction mechanisms between plasmonic NPs and the photoelectrodes with the goal of enhancing the limit-of-detection of PEC biosensors.
In order to create photoelectrodes that were fabricated using facile benchtop methods designed to enhance the photocurrent of PEC transducers, wrinkled scaffolds were used to fabricate photoelectrodes that show an order of magnitude enhancement in photocurrent compared to the planar electrodes. These electrodes were further used in label-free signal-off DNA biosensing without any amplification steps. Limit-of-detection of 200 times lower were reported using these wrinkled photoelectrodes, than planar electrodes.
Gold (Au) and TiO2¬ NPs were used as model materials to investigate the interaction between plasmonic and semiconductor NPs on a photoelectrode. The modulation of photocurrent was examined by varying the concentration of Au NPs and under different optical excitation wavelengths. UV light excitation provided larger photocurrent enhancement – at low concentration of Au NP – than visible light excitation. Furthermore, anodic photocurrent generation efficiencies by the photoelectrodes, which were prepared by using only Au NPs, were compared between interband and intraband excitation. The Au NP photoelectrodes demonstrated higher anodic photocurrent at interband excitation than intraband excitation and were further optimized by varying the size and deposition time of the Au NPs. Following this, Au NP- labeled DNA was used to study the effect of the distance between Au NPs and TiO2 NPs on the magnitude of the measured photocurrent. When Au NPs were in proximity with TiO2, they increased the generated photocurrent; however, they reduced the measured photocurrent when they were positioned further away from TiO2 NPs. Utilizing this switching behavior of PEC signals, a differential signal generation strategy was adopted to achieve a biosensor with enhanced sensitivity and signal-to-noise ratio.
Ultimately, we designed a PEC signal transduction strategy to detect nucleic acids without target labeling. In this assay, Au NP-labeled DNA was used as a signal-amplification-barcode that was introduced to the assay following target binding. This label-free PEC biosensor showed a low limit-of-detection (3 fM), broad (1 fM – 100 pM) linear range, and capability to detect single and double base-mismatched sequences of DNA. Thus, this work presents materials and signal transduction innovations that enhance the performance metrics of biosensors. / Dissertation / Doctor of Philosophy (PhD) / Detection and quantification of biomolecules is of utmost importance in early diagnosis, disease monitoring, prognosis, and disease management. In the past few decades, enormous efforts have been put towards utilizing photoelectrochemical (PEC) processes for biomolecular detection due to their high sensitivity. Gold nanoparticles are frequently being used to amplify the signal in the PEC bio-detection assay due to their plasmonic properties. However, the exact nature of the interaction between gold nanoparticles and the electrode material has not been determined. In this thesis, we investigated the interaction of gold nanoparticles with photoelectrode materials when they are separated by nucleic-acid sequences. Excitation energy and nucleic-acid length were varied to modulate the PEC current. The improved understanding of this interaction was further utilized to achieve a programmable response of nucleotide sensor from the photoelectrodes upon detecting the analyte of interest. We further developed different types of biosensing assay designs and examined their performance in terms of limit-of-detection, sensitivity, and specificity. Finally, we developed a new class of biosensor for detecting nucleic acids in bodily fluid and assessed the assay by using both electrochemical and PEC signal readout.
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FUNCTIONAL NUCLEIC ACIDS AS KEY COMPONENTS IN BIOSENSORSQian, Shuwen January 2023 (has links)
The functionality of nucleic acids beyond genetics has attracted more attention over the past decades. Functional nucleic acids (FNA), including aptamers and nucleic acid-based enzymes, are well-known for their target binding and reaction catalysis abilities. FNA can be obtained through a technology called in vitro selection, which allows the isolation of customized FNA for various applications. In particular, FNA have received much interest in biosensing application. Their wide range of sensing targets, intrinsic stability, and high specificity have qualified them as the molecular recognition element in biosensors.
This thesis explored the utilization of FNA to tackle real-world biosensing challenges, especially for pathogenic bacteria detection. The first project aimed to make the most use of in vitro selection to derive FNA that can meet the requirements of terminal applications. A few feasible approaches were proposed based on lessons from Mother Nature and validated by innovative scientist pioneers. In the second research project, I characterized an RNA-cleaving DNAzyme for Clostridium difficile infection diagnosis. This DNAzyme displayed high sensitivity and specificity for clinical C. difficile strains, making it a competitive candidate for a potential point-of-care diagnostic tool. In the next chapter, I incorporated a Legionella pneumophila-responsive RNA-cleaving DNAzyme into a bead-based assay for practical on-site detection. This assay exhibited a high stability and functionality in the cooling tower water samples, the real-world application environment. The following chapter was to optimize this assay further with a coupled rolling circle amplification strategy. This additional amplification speeded up the detection process, improved the limit of detection, and enabled the colorimetric results that are observable to the naked eye. These research aimed to advance the practical applications of FNA as key components in biosensors. I hope readers find this thesis insightful and inspirational for the development of the FNA field. / Thesis / Doctor of Philosophy (PhD)
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Developing Electrochemical Aptamer-based Biosensors for Quantitative Determination of Cyanotoxins in WaterVogiazi, Vasileia January 2020 (has links)
No description available.
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Measuring Individual Cell Cyclic Di-GMP: Identifying Population Diversity and Cyclic Di-GMP HeterogeneityMiller, Samuel I., Petersen, Erik 05 March 2020 (has links)
Cyclic di-GMP is a second messenger used by bacteria to regulate motility, extracellular polysaccharide production, and the cell cycle. Recent advances in the measurement of real time cyclic di-GMP levels in single cells have uncovered significant dynamic heterogeneity of second messenger concentrations within bacterial populations. This heterogeneity results in a wide range of phenotypic outcomes within a single population, providing the potential for population survival and adaptability in response to rapidly changing environments. In this chapter, we discuss some of the measurement technologies available for single-cell measurement of cyclic di-GMP concentrations, the resulting discovery of heterogeneous cyclic di-GMP populations, the mechanisms bacteria use to generate this heterogeneity, and the biochemical and functional consequences of heterogeneity on cyclic di-GMP effector binding and the bacterial population.
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