Дисертації з теми "Hydrogen Peroxide Biosensor"

The main hypothesis in this study is the development of a nanocomposite mediated amperometric biosensor for detection of hydrogen peroxide. The aim is to combine the electrochemical properties of both polyaniline and ferrocenium hexafluorophosphate into highly conductive nano composites capable of exhibiting electrochemistry in non acidic media
shuttling electrons between HRP and GCE for biosensor applications.

Hygroscopic insulator field effect transistors (HIFETs) are organic transistors with promising characteristics for biosensing applications. However, their fundamental sensing mechanisms are not yet fully understood. This thesis explores HIFET sensors through detailed electrical and optical characterisation, providing vital insights into the distinct mechanisms by which HIFETs detect biologically relevant chemicals. Hydrogen peroxide, a by-product of enzymatic reactions, oxidises the organic semiconductor, modulating the output current. Ionic solutions, such as KCl, NaCl and HCl, modulate the current by changing double layer capacitance. These insights are foundational for the continued development of HIFETs as effective multipurpose biosensing platforms.

A health condition called “Oxidative Stress” (OS), resulting from an excessive level of Reactive Oxygen Species (ROS) is a “state harmful to the body, which arises when oxidative reactions exceed antioxidant reactions because the balance between them has been lost”[1] OS appears to be associated with and might be a cause of, many serious diseases such as cardio-vascular accidents, cancer, Parkinson’s and Alzheimer’s[2]. This is not surprising as ROS are free oxygen radicals that can attack lipids, proteins, cellular membranes, enzymes and even modify DNA. Extensive correlation studies have shown that the complex impedance spectrum of blood samples from patients diagnosed with an OS syndrome differs significantly from the spectra obtained from the blood of healthy people, which is quite normal as the presence of an excessive amount of ROS should affect the physico-chemical properties of a blood sample. Measuring the complex impedance spectrum of a blood sample can be done quickly by means of low-cost electronic devices, making possible and affordable the early detection of OS among a large population. In order to quantitatively evaluate the OS, the impedance spectra being insufficient, the concentration of oxidative stress markers such as hydrogen peroxyde, malondialdehyde or F2 isoprostanes needs to be measured. Such measurements can, for instance, be used for monitoring the severity of a disease during a treatment. These concentration measurements are traditionally based upon analytical techniques but recently biosensors acting as transducers transforming directly a specific biochemical reaction into a measurable signal have been developed. They are essentially obtained by modifying the surface of metal or carbon electrodes using biomaterials such as enzymes antibodies or DNA that allow bindings or catalytic reactions with other specific biomaterials to occur on the surface of the electrodes. The resulting modifications of the electrical properties of the medium separating the electrodes can be analyzed through ad-hoc electronic and signal processing systems to yield the desired concentration. Biosensors have the advantages of rapid analysis, low-ost and high-precision. They are widely used in various fields, such as medical care, disease diagnosis and food analysis [3]. Hydrogen peroxide (H2O2) generated by cellular processes directly via two-electron reduction of molecular oxygen or indirectly via dismutation of superoxide, is the most widely studied ROS and its overproduction results in OS. Therefore, an ability to quantify the level of hydrogen peroxide and by ricochet the assessment of oxidative stress can be useful in order to assess certain health conditions occurring inside the body and as a result, an integrated electrochemical biosensor coupled with the hydrogen peroxide quantification can become a practical solution as a point of care device at home[4] Most of the time, H2O2 biosensors are based on HRP (Horseradish peroxidase) which is the most commonly used enzyme in the design of biosensors that can supervise the activity of oxidases and determine in terms of concentration, oxidase substrate such as lactate oxidase, cholesterol oxidase, or glucose oxidase, which all induce the production of hydrogen peroxide (HRP’s substrate). In the first part of this research, we explore the development of low-cost and compact measurement systems aiming to determining the impedance of biological samples as they grant access to information from electrical cellular characteristics. It is indeed possible to measure capacitance or conductance that are dependent on the health state of cells. The development of such measurement systems allowing the portability of biological essays requires sensitive electronics. Afterward, in the second part of our work, we explore the design of an electrochemical biosensor by immobilizing an enzyme (HRP) onto the surface of golden electrodes in order to detect and assess the analyte, hydrogen peroxide (H2O2). We also discuss the design of a potentiostat readout circuit to measure and convert the biosensor’s current. The combined results of the two parts of this work can be considered as a first prototype of a low cost and robust instrument easy to use in the field, away from a biological laboratory, with the goal of reaching the so called “point of care diagnostic” [5] The present thesis is organized as follows: Chapter I, introduces the present thesis. In Chapter II, we provide an overview in the field of biosensing technology. Chapter III deals with the design of a portable EIS measurement system to investigate reactive oxygen species in blood. Chapter IV presents an improved version of the previously designed instrument. Moreover, it points out the significance of EIS-based blood analysis through relevant medical diagnosis parameters such as hematocrit and erythrocyte sedimentation rate, extracted from the measured impedance spectra. In Chapter V we discuss on one hand the design of the H2O2 biosensor, and on the other hand the realization of the front-end circuit of the amperometric sensor. Finally, in Chapter VI, a conclusion is drawn..

The main purpose of this work is study of electrochemical hydrogen peroxide and ascorbate reactions on electrodes modified by Prussian blue (PB), with the aim to apply these electrodes in creation of sensors and biosensors. For this purpose, a detailed study of electrochemical reduction of hydrogen peroxide, as well as of oxidation of ascorbate at Prussian blue modified rotating disk electrode. In view of the results obtained, a mechanism for hydrogen peroxide reduction at PB modified electrode has been proposed. In accordance with this mechanism, electron transfer appears to be rate-limiting step. The kinetics of decomposition of PB modified electrode in the course of a cathodic reduction of hydrogen peroxide has been studied, and the influence of different factors to this process has been determined. Prototypes of sensors and biosensors, for different analytes have been elaborated and tested.
Darbo tikslas yra elektrocheminių vandenilio peroksido ir askorbato reakcijų tyrimas ant Berlyno mėlynuoju (BM) modifikuotų elektrodų, siekiant pritaikyti šiuos elektrodus jutiklių ir biojutiklių kūrimui. Ištirta vandenilio peroksido redukciją ir askorbato oksidaciją naudojant sukamojo disko elektrodą. Gauti rezultatai galimai įrodo stadijinį vandenilio peroksido katodinės redukcijos mechanizmą vykstantį ant BM modifikuoto elektrodo. Detaliai ištirta BM sluoksnio irimo kinetika vandenilio peroksido elektroredukcijos metu, ir nustatyti faktoriai, įtakojantys irimo proceso greitį. Sukurti jutiklių ir biojutiklių prototipai, kurie galėtų būti panaudoti biologiškai aktyvių medžiagų (vandenilio peroksido, askorbato, gliukozės) nustatymui.

Darbo tikslas yra elektrocheminių vandenilio peroksido ir askorbato reakcijų tyrimas ant Berlyno mėlynuoju (BM) modifikuotų elektrodų, siekiant pritaikyti šiuos elektrodus jutiklių ir biojutiklių kūrimui. Ištirta vandenilio peroksido redukciją ir askorbato oksidaciją naudojant sukamojo disko elektrodą. Gauti rezultatai galimai įrodo stadijinį vandenilio peroksido katodinės redukcijos mechanizmą vykstantį ant BM modifikuoto elektrodo. Detaliai ištirta BM sluoksnio irimo kinetika vandenilio peroksido elektroredukcijos metu, ir nustatyti faktoriai, įtakojantys irimo proceso greitį. Sukurti jutiklių ir biojutiklių prototipai, kurie galėtų būti panaudoti biologiškai aktyvių medžiagų (vandenilio peroksido, askorbato, gliukozės) nustatymui.
The main purpose of this work is study of electrochemical hydrogen peroxide and ascorbate reactions on electrodes modified by Prussian blue (PB), with the aim to apply these electrodes in creation of sensors and biosensors. For this purpose, a detailed study of electrochemical reduction of hydrogen peroxide, as well as of oxidation of ascorbate at Prussian blue modified rotating disk electrode. In view of the results obtained, a mechanism for hydrogen peroxide reduction at PB modified electrode has been proposed. In accordance with this mechanism, electron transfer appears to be rate-limiting step. The kinetics of decomposition of PB modified electrode in the course of a cathodic reduction of hydrogen peroxide has been studied, and the influence of different factors to this process has been determined. Prototypes of sensors and biosensors, for different analytes have been elaborated and tested.

Orientador: Lauro Tatsuo Kubota
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica
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Resumo: Neste trabalho foram investigados diferentes métodos de imobilização da enzima HRP empregando como matrizes as monocamadas auto-organizadas formadas sobre eletrodos de ouro, bem como a avaliação da influência do processo de imobilização do elemento biológico no desempenho analítico do biossensor. Para isso. as monocamadas utilizadas foram formadas por meio de tióis com diferentes estruturas, tamanho de suas cadeias carbônicas e grupos terminais. Foi possível constatar que o tamanho da cadeia carbônica de um tioI influencia especialmente no empacotamento da monocamada e, conseqüentemente, na eficácia da imobilização das biomoléculas. Pelos estudos realizados visando a caracterização das SAM sobre a superfície eletródica foi possível verificar que os tióis que possuem em sua cadeia um número menor de carbonos (< 9) tendem a formar monocamadas com uma quantidade considerável de defeitos na superfície do ouro, o que leva a um recobrimento mais baixo. No entanto, os tióis que contém um número mais elevado de carbonos na cadeia apresentam um grau de recobrimento mais elevado e, no entanto, não são boas matrizes para biossensores eletroquímicos, pois podem passivar a superfície, diminuindo a transferência de elétrons e, como conseqüência, a sensibilidade do eletrodo. Quanto a imobilização da enzima nos eletrodos de ouro, verificou-se, por diferentes técnicas, que as monocamadas que possuem grupo terminal -NH2 foram aquelas que proporcionaram os melhores resultados, provavelmente devido ao uso do glutaraldeído como ligante no processo de imobilização. Ao analisar adicionalmente o desempenho do biossensor para a determinação de peróxido de hidrogênio, verificou-se que a SAM formada pela cisteamina é a mais adequada para a imobilização da HRP, por propiciar tanto uma melhor eficácia na adsorção enzima quanto uma sensibilidade mais elevada para H2O2
Abstract: In this work different immobilization procedures of HRP were investigated using as support mIatrices the self-assembled monolayers formed on gold electrodes, as well as the evaluation of the influence of these immobilization processes in the biosensor performance. For this, the used monolayers were prepared by thiols with different structures, carbon chains size and terminal groups. It was possible to have evidence that the thiol carbon chain size influences especially in the coverage monolayer and, consequently, in the efficiency of the biomolecule immobilization. From the studies carried out for the SAM characterization on the electrode surface it was possible to verify that thiols with smaller chain (n<9) trends to form monolayers with a considerable amount of defects on gold surface, that it leads to a lower coverage. However, the thiols with a higher carbon chain present a higher coverage degree, are not being good matrices for electrochemical biosensors, because it can passive the surface, making difficult the electron transfer and, consequently, the electrode sensitivity. In relation to the enzyme immobilization on gold electrodes it was verified, for different techniques, that monolayers that possess -NH2 terminal group provided the best results, probably due to the use of glutharaldeyde as ligant at the immobilization process. Analyzing the biosensor performance for the hydrogen peroxide determination was verified that SAM formed by cysteamine is more adequate for HRP immobilization, because provide the better efficiency in the enzyme immobilization associated to high sensitivity for H2O2
Doutorado
Quimica Analitica
Doutor em Ciências

Os desafios atuais no desenvolvimento de biossensores abrangem diversos aspectos, tais como a necessidade de se aperfeiçoar a interface de contato entre o substrato e o material biológico, a eficiência de transdução do sinal químico em um sinal mensurável, o tempo de resposta, a compatibilidade dos biossensores com matrizes biológicas e a integração de diferentes elementos de reconhecimento biológico em um único dispositivo, visando a detecção de distintos analitos. Nesse contexto, o desenvolvimento da nanociência tem criado recursos bastante atraentes para otimizar os aspectos descritos acima. O presente trabalho apresenta, portanto, estudos realizados para a construção de mediadores nanoestruturados que possam operar de maneira mais eficiente que os correspondentes materiais maciços (sistemas não-nanoestruturados). Em uma das abordagens utilizadas, um mediador híbrido de hexacianoferrato de cobre/polipirrol (CuHCNFe/Ppy) teve suas propriedades eletroquímicas aliadas às propriedades morfológicas e eletrônicas de um feltro revestido com nanotubos de carbono do tipo \"cup-stacked\" (feltro/NTCCS) para o desenvolvimento de um sensor de H2O2. O feltro/NTCCS é uma malha hidrofílica condutora que permite uma dispersão bastante uniforme do mediador híbrido. Essa característica, aliada ao aumento da área eletroativa e à interação eletrônica existente entre o CuHCNFe/PPy e os nanotubos de carbono criaram uma plataforma favorável para a construção de um biossensor de glicose. Em uma segunda estratégia, esferas de poliestireno com diâmetros de 300, 460, 600 e 800 nm foram utilizadas como molde para a formação de filmes de CuHCNFe/PPy macroporosos. Os distintos mediadores foram aplicados na detecção de H2O2 com o intuito de se correlacionar a importância do tamanho do poro com o desempenho analítico obtido. Diferentemente do esperado, os mediadores maciços e porosos apresentaram desempenhos analíticos bastante similares, o que levou a uma consideração das propriedades termodinâmicas de superfícies curvas, da molhabilidade de materiais porosos e da influência da cinética eletroquímica na utilização de sistemas porosos. Tais plataformas também foram aplicadas com sucesso na construção de biossensores de glicose e de colina. Por fim, foi possível sintetizar mediadores nanoestruturados através da imobilização de camadas de azul da Prússia e de CuHCNFe dentro das cavidades de filmes de TiO2 mesoporosos (13, 20 e 40 nm de diâmetro). Os resultados obtidos demonstraram a possibilidade de se modular o desempenho dos sensores de H2O2 em função do diâmetro dos poros e da quantidade de mediador imobilizado. A união dos resultados analíticos obtidos com os dados de microscopia eletrônica de varredura possibilitou observar a importância do efeito de confinamento no desempenho dos mediadores. Além disso, dados espectroscópicos na região do visível foram fundamentais para relacionar a presença de defeitos estruturais com a reatividade do material. No fim, tais plataformas foram utilizadas para a formulação de biossensores de colina.
Nowadays, the challenges in the development of biosensors cover various aspects such as the need to improve the interface between the substrate and the biological material, the efficiency of the chemical signal transduction in a measurable one, the response time, the compatibility with biological matrices and the integration of different biological recognition elements in a single device, in order to perform detections of different analytes. In this context, the development of nanoscience has created very attractive features to optimize the aspects described above. Consequently, the present work studies the build up of nanostructured transducers that can operate more efficiently than the corresponding bulk materials (systems non-nanostructured). In one of the approaches used, a hybrid transducer consisting of copper hexacyanoferrate/polypyrrole (CuHCNFe/Ppy) had its electrochemical properties combined with the morphological and electronic properties of a felt decorated with cup-stacked type carbon nanotubes (felt/CSCNT) for development of a H2O2 sensor. Felt/CSCNT is a hydrophilic conductive mesh that allows a uniform dispersion of the hybrid transducer. This feature, coupled with the improvement of electroactive surface and with the electronic interaction among the CuHCNFe/Ppy and carbon nanotubes have created a favorable platform for the construction of a glucose biosensor. In a second strategy, polystyrene spheres with diameters of 300, 460, 600 and 800 nm were used as templates for the formation of macroporous CuHCNFe/Ppy films. The transducers were used to detect H2O2 in order to correlate the importance of pore size with the obtained analytical performance. Unlike expected, porous and bulk transducers presented very similar analytical performances, which led to a consideration of the thermodynamic properties of curved surfaces, the wettability of porous materials and the influence of electrochemical kinetics during the use of porous systems. Such platforms have also been successfully applied in the preparation of glucose and choline biosensors. Finally, it was possible to synthesize nanostructured transducers through the immobilization of Prussian blue layers and CuHCNFe inside the cavities of mesoporous TiO2 films (pore diameters of 13, 20 and 40 nm). The obtained results demonstrated the possibility of modulating the performance of H2O2 sensors according to the pore diameter and the amount of immobilized transducer. The union of the obtained analytical results with scanning electron microscopy data showed the importance of confinement effect on the transducers performances. In addition, spectroscopic data in the visible region were essential to correlate the presence of structural defects with the material reactivity. In the end, these platforms were used for the formulation of choline biosensors.

Os biossensores são dispositivos empregados para a detecção de um analito específico, podendo assim ser no controle de qualidade nos alimentos para determinar a presença de micro-organismos, toxinas ou metabólitos. O presente estudo objetiva desenvolver um biossensor condutométrico, baseado na imobilização de peroxidasse em membranas de quitosana e quitosana com nanopartículas de ouro (AuNP) para a detecção de peroxido de hidrogênio. O trabalho foi dividido em três etapas. Na primeira etapa foi estudada a obtenção de AuNP empregando agentes redutores biológicos, sendo avaliados três monossacarídeos (glicose, frutose e galactose), três dissacarídeos (sacarose, maltose e lactose), dois biopolímeros (amido e quitosana), assim como os extratos obtidos a partir das folhas de hortelã (Mentha piperita) e cascas de furtas de abacaxi (Ananas comosus), banana (Musa sp. ), maracujá (Passiflora edulis), tangerina (Citrus reticulata). A quitosana mostrou-se como o melhor agente redutor na síntese das AuNP, as quais foram empregadas na segunda etapa para a produção de membranas. Três tipos de membranas foram processadas, membranas de quitosana sem AuNP e membranas de quitosana com AuNP com concentrações de 8 e 11mM., as quais foram caraterizadas morfológica e eletricamente. Finalmente foi avaliada a imobilização da peroxidasse usando quatro tratamentos diferentes, sendo a dispersão da peroxidasse nas soluções filmogênicas precursoras das membranas a mais eficiente. A resposta elétrica destas membranas é dependente da concentração de AuNP e da presença de enzimas, e também foi alterada quando as mesmas foram expostas a soluções de tampão fosfato com diferentes concentrações de peroxido de hidrogênio. Isto constitui o principio de operação dos biossensores condutométricos desenvolvidos neste trabalho.
Biosensors are devices used for detecting a specific analyte, and thus can be used in quality control of food for determining the presence of micro-organisms, toxins or metabolites. The present study aims to develop a conductometric biosensor based on the immobilization of peroxidase in membranes of chitosan and chitosan with gold nanoparticles (AuNP) for the detection of hydrogen peroxide. The work was divided into three stages. In the first stage, methods for obtaining AuNP employing biological reducing agents were studied, evaluating three monosaccharides (glucose, fructose and galactose), three disaccharides (sucrose, maltose and lactose), two biopolymers (starch and chitosan), as well as the extracts obtained from the leaves of mint (Mentha piperita) and husks dost thou pineapple (Ananas comosus), banana (Musa sp), passion fruit (Passiflora edulis), mandarin (Citrus reticulata). Chitosan exhibited the best behavior as reducing agent for the synthesis of AuNP, which were employed in the second step for the production of membranes. Three types of membranes were processed, chitosan membranes without AuNP and chitosan membranes with AuNP with concentrations of 8 and 11mM, which were morphologically and electrically characterized. Finally the peroxidase immobilization was evaluated using four different procedures, being the dispersion of the peroxidase in filmogenic solutions precursor of membranes the more efficient. The electrical response of these membranes, depends on the AuNP concentration and the presence of enzymes, and was also altered when they were exposed to hydrogen peroxide containing phosphate buffer solutions. This constitutes the principle of operation of the conductometric biosensor developed in this work.

碩士
國立臺灣科技大學
化學工程系
100
This study was comprised of two parts: (I) the synthesis of trisoctahedral gold nanocrystal (AuNC) and core-shell AuNCs@Pt catalysts for the applications of electrochemical sensing of hydrogen peroxide; (II) the immobilization of enzymatic species by adding cross-linking agent to fabricate electrochemical glucose sensor. For the first part, the surface morphology and crystal lattice of the synthesized nano-metallic catalysts were investigated using SEM, XRD. The synthesized AuNCs showed many active sites due to their polyhedral structure. In order to enhance the catalytic ability, the AuNCs with outer layer of platinum (to form polyhedral nano platinum crystals) was synthesized which provides a particular advantage of only a small amout of platinum was needed. The results of UV-vis spectropy and electrochemical acid treatment showed that the bimetallic catalyst is core-shell structure which was almost completely covered by reduced platinum. The detection of hydrogen peroxide was measured by electrochemical methods. Moreoer, the optimized parameters for electrochemical analyses including the applied voltage and the surface protection layer were applied for the detection of hydrogen peroxide. The results showed that the detection limit of 10 μM, with a linear range of detection from 0.01 to 5.1 mM (R2=0.997), high sensitivity of 397.37 μA/(mMcm2), and excellent anti-interfering ability were obtained for the prepared sensing system. For the second part, the prepared sensing layer on the electrodes was further applied for the detection of glucose. In this study, the synthesized AuNCs were used to adsorb enzyme molecules which were followed by the addition of cross-linking agent to ensure the combination. The enzyme loading and operating parameters were optimized. It showed that the assembled sensor prepared under the optimized condition provided mild environment for enzyme immobilization and facilitated the bio-reaction between enzyme and bio-species, which allowed enzyme exhibiting good affinity and stability. The obtained glucose sensing at 0.5 V v.s Ag/AgCl applied potential showed linear range of 1.0 to 7.0 mM (R2=0.996), with sensitivity of 86.93 μA/mMcm2. We have shown that a highly sensitive glucose biosensor with good reproducibility and precision, high sensitivity, and great stability was successfully prepared.

Title: Study of cerium oxide thin films for biosensing applications Author: Yuliia Kosto Department: Department of Surface and Plasma Science Supervisor: Prof. RNDr. Vladimír Matolín, DrSc. Abstract: The presented scientific work was conducted in two main directions. The first one is an investigation of the simple biomolecules (glycine and sarcosine) bonding to cerium oxide model films by surface science techniques: photoelectron and near-edge X-ray absorption spectroscopies. Adsorption chemistry and thermal stability of the molecules on the oxides were studied in relation to the oxidation state of ceria cations, film morphology, and molecular deposition method. The oxygen vacancies in the oxide were shown to affect the adsorption geometry of glycine and stimulate molecular decomposition. The polycrystalline oxide morphology provided stabilizing effect on the glycine adlayer. Sarcosine deposited in vacuum formed densely packed adlayer with the molecules directed outwards. Interestingly, the results revealed that molecular film deposited from the aqueous solution, in contrast to deposition in vacuum, induces continuous reduction of the cerium oxide during thermal annealing. The second part is a study of polycrystalline cerium oxide thin films as an electrode for electrochemical and electrochemiluminescent...

碩士
淡江大學
化學學系碩士班
102
In current study, a cobalt oxide based uric acid biosensor is fabricated through a drop coating of glutaraldehyde, and uricase onto a BSA coated Co3O4 based rotating disc graphite electrode (RGDE). Uric acid is measured via an uricase based Co3O4 modified biosensor cathodicly. The amperometric signal of this sensor is measured based on the uricase converts uric acid into hydrogen peroxide and reduces by Co3O4. The homemade cobalt oxide fabricated by hydrothermal synthesis is better than the commercial one. Qualitative analysis and morphology of cobalt oxide was characterized by High resolution X-ray diffractmeter (HRXRD) and scanning electron microscopic (SEM). The results show that homemade cobalt oxide is Co3O4 and exhibits rectangular sheets with pore and piled the nanosheets of each other. The uric acid biosensor was simply fabricated with mixing carbon ink and 70% Co3O4 then placed on RDGE and dried in the oven at 80℃ for an hour. Followed drop coated 5 μL 0.5 % BSA, 5 μL 0.1% glutaraldehyde, and 5 μL 0.5 units uricase onto electrode till dried in 4℃ refrigerator. The biosensor showed optimum conditions for the analysis of uric acid are in 0.05 M pH 9.5 Clark & Lubs buffer, when applied at 0.05 V (vs. Ag/AgCl) with rotating rate of 400rpm. The linear range of this scheme covers from 2 μM to 102 μM (R=0.999), with sensitivity is 18.24 μA/mM and a detection limit of 0.6 μM. The relative standard deviation (RSD) for 20 times successive measurement of 25 μM UA is 1.38%, and the response time (t90%/10%) is 27.34 s. These metabolites have interference except acetaminophen, creatine, and dopamine. Others ratio of interference were between -556.56 % and 5.8%. Among these metabolites, ascorbic acid has -556.56 % interference. Therefore, the removal of AA interference could be eliminated by pretreatment of 10 units ascorbate oxidase with sample for one hour in advance. This sensor has good stability during 83 days study with 4.12% of RSD.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
As espécies reativas de oxigénio são um grupo de moléculas derivadas do oxigénio, que são formadas por redução-oxidação (redox) ou por excitação eletrónica, e que têm diferentes distribuições temporais e espaciais e uma ampla gama de concentrações intracelulares e extracelulares. O peróxido de hidrogénio é umas destas espécies com mais interesse ao nível da sinalização redox celular sendo que as suas concentrações podem desencadear diferentes respostas, existindo níveis limiares que separam o estado patológico do estado fisiológico e por conseguinte, o estado de oxidative eustress do estado de oxidative distress. Assim, é importante compreender como é que esta molécula se comporta e encontra no corpo humano, por forma a realizar a sua deteção e monitorização. Atualmente, são aplicados vários métodos analíticos destacando-se os espectroscópicos (quimioluminescência e fluorescência) e os eletroanalítcos (sensores e biossensores). Os biossensores, embora ainda apresentem algumas desvantagens assumem-se como um dos maiores desafios na deteção e monitorização do peróxido de hidrogénio utilizando uma molécula de reconhecimento biológico ou mediadores de transferência de eletrão tais como os hexacianoferratos, conhecidos como “peroxidases artificiais”. Estes sensores são capazes de fornecer informações sobre a dinâmica de concentração em tempo real, que não é acessível com outros métodos. Torna-se, então, claro que o H2O2 desempenha funções fundamentais nometabolismo sendo a compreensão do seu papel biológico bastante relevante e os métodosde deteção e monitorização um enorme desafio. Assim, foram aplicadas diversas metodologias analíticas para compreensão da sua bioatividade no tecido cerebral, em células cancerígenas e outras células.
Reactive oxygen species are a group of molecules derived from oxygen, which are formed by reduction-oxidation (redox) or by electronic excitation, and which have different temporal and spatial distributions and a wide range of intracellular and extracellular concentrations. Hydrogen peroxide is one of this species interesting reactive oxygen species in terms of cellular redox signalling and its concentrations result in different responses. Different levels of hydrogen peroxide separate the pathological state from the physiological state and, the oxidative eustress state from the oxidative distress state. Thus, it is important to understandhow this molecule behaves and finds in the human body to carry out its detection and monitoring. Nowadays, several analytical methods are applied, highlighting spectroscopic(chemiluminescence and fluorescence) and electroanalytical (sensors and biosensors).Biosensors, although they still have some disadvantages, are one of the biggest in challenges in detection and monitoring of hydrogen peroxide using a biological recognition molecule or electron mediators such as hexacyanoferrate compounds known as an “artificial enzyme peroxidase”. This type of sensors can provide information regarding the real-time concentration dynamics of hydrogen peroxide, that are not accessible to other methods. It then becomes clear that hydrogen peroxide plays key roles in metabolism being the understanding of its biological role very relevant and the methods detection and monitoring a huge challenge. Thus, several analytical methodologies were applied to understand its bioactivity in brain tissue, cancer cells and other cells.

Niniejsza praca poświęcona jest opracowaniu bioelektrod do enzymatycznego oznaczania stężenia neuroprzekaźników, tlenu oraz nadtlenku wodoru w zintegrowanym układzie sensorowym, zasilanym enzymatycznym bioogniwem. Zaprojektowane biourządzenie wykorzystywało łączność bezprzewodową do transferu wyników pomiarowych z czujnika bezpośrednio do komputera. Celem badań było skonstruowanie nieinwazyjnego i taniego układu z możliwością jego łatwej i samodzielnej obsługi.Do elektrochemicznego oznaczania nadtlenku wodoru posłużył bienzymatyczny czujnik - elektroda węglowa pokryta nanorurkami węglowymi, skutecznie wiążącymi dwa enzymy: katalazę oraz lakazę (lub oksydazę bilirubinową). Zadaniem pierwszego z enzymów, katalazy była kataliza rozkładu nadtlenku wodoru do tlenu, natomiast drugi enzym, lakaza (lub oksydaza bilirubinowa) redukowała tlen do wody w procesie czteroelektronowym. Jednoczesne zastosowanie dwóch enzymów umożliwiło wyznaczenie całkowitego stężenia nadtlenku wodoru, przez ominięcie problemów wynikających z niestabilności analitu. Zaproponowana została metoda zwiększająca efektywność przygotowanej warstwy katalitycznej. Polega ona na nanostrukturyzacji podłoża elektrodowego poprzez osadzenie lasu nanorurek węglowych modyfikowanych grupami naftylowymi.
This work is devoted to the development of bioelectrodes for the enzymatic determination of the concentration of neurotransmitters, oxygen and hydrogen peroxide in an integrated sensor system powered by an enzymatic biofuel cell. The designed biosensing device used wireless communication to transfer the measurement results from the sensor directly to the computer. The aim of the research was to construct a non-invasive and cheap system with the possibility of its easy and independent operation.The electrochemical determination of hydrogen peroxide was performed by a bienzymatic sensor - a carbon electrode covered with carbon nanotubes, which were effectively binding two enzymes: catalase and laccase (or bilirubin oxidase). The task of the first enzyme, catalase was to catalyze the decomposition of hydrogen peroxide to oxygen, while the second enzyme, laccase (or bilirubin oxidase) reduced oxygen to water in a four-electron process. The simultaneous use of two enzymes allowed to determine the total concentration of hydrogen peroxide, bypassing the problems resulting from instability of the analyte. A method for increasing the efficiency of the prepared catalytic layer was proposed. It involves the nanostructuring of the electrode substrate by embedding a forest of carbon nanotubes modified with naphthyl groups.

碩士
南台科技大學
化學工程與材枓工程系
95
ABSTRACT Nowadays, sometimes the hydrogen peroxide is used in the food industry for the purpose of preservation and therefore, developing a hydrogen peroxide sensor is an important research subject. In recent years, the diabetes has become one of the top ten causes of death for the people in our country. Therefore, developing a glucose biosensor which can detect the glucose rapidly and conveniently is also an important research subject. The glucose and oxygen can be catalyzed by the glucose oxidase to produce the gluconic acid and hydrogen peroxide. A study was conducted to use the ferrocene to modify the carbon paste electrode. Because the ferrocene(Fe(C5H5)2) possessed the excellent catalytic characteristic, it can be used with the graphite carbon powders which possessed the excellent conductivity to make the carbon paste electrode and to elevate the responding current of hydrogen peroxide. The responding current of hydrogen peroxide is detected in the phosphate buffer solution(PBS) and then the concentration of the hydrogen peroxide can be obtained and consequently, the concentration of the glucose can be determined. The experimental design is used widely in many fields and it is an important tool for the test and the improvement of the process of industrial production. A study of factorial design involving three factors (operating potential, stirring rate, and pH value of PBS) and two levels was performed to analyze the effect of reaction parameters on the sensitivity and average responding current of detection of hydrogen peroxide for the carbon paste electrode modified with the ferrocene. The results of the calculating the effects showed that the sensitivity and average responding current at -200 mV operating potential were higher than those at -50 mV operating potential, the sensitivity and average responding current at 500 rpm stirring rate were higher than those at 300 rpm stirring rate, and the sensitivity and average responding current at pH=7.4 were higher than those at pH=4.0. The SPSS(Statistical Package for the Social Science) was used to perform the analysis of variance. The results showed that the main effect of operating potential ( F = 151.494, p value ＜ 0.05 ) , the main effect of stirring rate ( F = 7.288, p value ＜ 0.05 ) and the main effect of pH value ( F = 94.461, p value ＜ 0.05 ) were significant on the sensitivity. The main effect of operating potential ( F = 141.515, p value ＜ 0.05 ) , the main effect of stirring rate ( F = 7.487, p value ＜ 0.05 ) and the main effect of pH value ( F = 89.901, p value ＜ 0.05 ) were also significant on the average responding current. The results showed that the optimum operating condition for this research is operating potential = -200 mV , stirring rate = 500 rpm and pH = 7.4. At 30℃, -200 mV operating potential, 500 rpm stirring rate and in 0.05M PBS buffer solution ( pH = 7.4 ) , when the carbon paste electrode was modified with the ferrocene [ferrocene : graphite carbon powder = 3 : 7 ( weight ratio )] , the detection limit was 0.02 mM H2O2 , the linear range was 0.02～1.2 mM H2O2 , R2 = 0.9998 and the sensitivity was 161.43µA/cm2．mM H2O2.

碩士
南台科技大學
化學工程與材枓工程系
95
Due to an occasional use of hydrogen peroxide in the food industry for the purpose of preservation nowadays, a rapid and convenient sensor for detecting the hydrogen peroxide is an important research subject. In recent years, diabetes has become one of the top ten causes of death for the people in our country and therefore, a convenient and rapid glucose biosensor also has become an important research subject. The glucose and oxygen can be catalyzed by the glucose oxidase and the glucose is oxidized to gluconic acid and the oxygen is reduced to hydrogen peroxide. A study was conducted to use the Coprecipitation method to prepare the Copper(Ⅱ) Hexacyanoferrate(Cu(Ⅱ)HCF) [Cu(Ⅱ)HCF : graphate carbon powders = 3 : 7(weight ratio)]. The Cu(Ⅱ)HCF was used to modify the carbon paste electrode because the Cu(Ⅱ)HCF possessed the excellent catalytic characteristic and it could be used with the graphite carbon powders and carbon paste to make the carbon paste electrode to elevate the responding current of the hydrogen peroxide. A study of factorial design involving three factors[(A)operating potential, (B)stirring rate, (C)pH value of phosphate buffer solution(PBS)] and two levels was performed to analyze the effect of reaction parameters on the sensitivity and average responding current of detection of the hydrogen peroxide for the carbon paste electrode modified with the Cu(Ⅱ)HCF. The analysis of variance was performed by the SPSS (Statistical Package for the Social Science). The results of the calculating the effects showed that the sensitivity and average responding current at -200 mV operating potential were higher than those at -50 mV operating potential and the sensitivity and average responding current at 500 rpm stirring rate were higher than those at 300 rpm stirring rate. The value of the effect of pH value was negative which showed that the sensitivity and average responding current at pH=7.4 were lower than those at pH=4.0. The results of analysis of variance showed that the main effect of operating potential(F =27.691, p value < 0.05)and the main effect of pH value(F =23.857, p value < 0.05)were significant on the sensitivity and the main effect of operating potential(F =23.053, p value < 0.05)and the main effect of pH value (F =19.312, p value < 0.05) were also significant on the average responding current. At -200 mV operating potential, 500 rpm stirring rate and in 0.05M PBS buffer solution(pH=4.0), the detection limit was 0.02 mM, the linear range was 0.02~2.8 mM H2O2, R2=0.9982 and the sensitivity was 141.04 μA/cm2ּmM H2O2. At -200 mV operating potential, 500rpm tirring rate and in 0.05M PBS buffer solution(pH=7.4), the detection limit was 0.02 mM, the linear range was 0.02~2.6 mM H2O2, R2=0.9945 and the sensitivity was 121.606 μA/cm2ּmM H2O2. The results showed that the optimum operating condition for this research is operating potential= -200mV , stirring rate = 500rpm, and pH = 7.4 .

博士
東海大學
化學工程與材料工程學系
97
In this study, the PANI or PANI(BSA) films was electrochemically synthesized on the Pt/Al2O3 base to form the PANI/Pt/Al2O3 or PANI(BSA)/Pt/Al2O3 electrode, and horseradish peroxidase (HRP) was then immobilized in PANI or PANI(BSA) films to construct the GA-HRP/PANI/Pt/Al2O3 or GA-HRP/PANI(BSA)/Pt/Al2O3 biosensor. Furthermore, the mesoporous silica SBA-15 was prepared and was employed to entrap HRP in order to construct a GA/SBA-15(HRP)/PANI/Pt/Al2O3 electrode. The properties and the performances of those bio-electrodes were fully investigated. Moreover, the influence of the dissolved oxygen on sensing of hydrogen peroxide with those PANI modified electrode was discussed and the possible elimination was proposed by using oxygen scavengers. For electrodes, such as GA-HRP/PANI/Pt/Al2O3 and GA-HRP/PANI(BSA)/Pt/Al2O3, the linear correlation for sensing hydrogen peroxide in a 0.1 M phosphate buffer were obtained in the range of 10 μM ~ 23.9 mM and 10 μM ~ 35.2 mM, respectively, while the sensitivities were 37.55 and 44.31μA mM-1, respectively. By comparing the stability after sensing 2.39 mM hydrogen peroxide for twenty-one times in a 17-day period, the percentage of residual response current were 40.8 % for PANI/Pt/Al2O3 and 80.5 % for PANI(BSA)/Pt/Al2O3, indicating that BSA was useful for improving the sensitivity and stability of PANI modified hydrogen peroxide biosensor. In addition, 0.5 mM of uric acid, urea, or ascorbic acid would cause less than 10% reduction of cyclic voltammetry (CV) current on sensing 2.39 mM hydrogen peroxide for GA-HRP/PANI/Pt/Al2O3. For the electrode GA/SBA-15(HRP)/PANI/Pt/Al2O3, the linear correlation and the sensitivity for sensing hydrogen peroxide sensor in a 0.1 M phosphate buffer were obtained in the range of 10 ~ 23.9 mM and 25.05 μA mM-1, respectively. After sensing 1.96 mM hydrogen peroxide for fifteen times in a 16-day period, the percentage of residual response current were 75.2 %, which was better than that of GA-HRP/PANI/Pt/Al2O3 (40.5%), implicating that SBA-15 was also able to stabilize the entrapped HRP and therefore improved the performance of the constructed biosensor. Finally, we demonstrated the influence of the dissolved oxygen on sensing hydrogen peroxide with PANI modified electrodes, and we proposed strategies to eliminate the influences. Our results inindicated that the oxygen scavenger such as sodium thiosulfate was able to effectively remove the dissolved oxygen, thereby reduce its effect on the performance of electrode, Meanwhile, sodium thiosulfate displayed the negligible effect on hydrogen peroxide measurements while its applied concentration was below 1 mM.

碩士
南台科技大學
化學工程與材枓工程系
94
ABSTRACT In recent years, diabetes has become one of the top ten causes of death for people in our country. Therefore, a rapid and convenient glucose biosensor becomes an important research subject. Nowadays, sometimes the preservatives are used in the food industry for the purpose of food preservation. Therefore, a hydrogen peroxide sensor also becomes an important research subject. The glucose and oxygen can be catalyzed by the glucose oxidase and the glucose is oxidized to gluconic acid and the oxygen is reduced to hydrogen peroxide. The electrode releases the electrons at the reductive potential and converts the mediator to the reductive state. Then the mediator at the reductive state releases the electrons to reduce the hydrogen peroxide to water and consequently the mediator at the reductive state is converted to the oxidizing state. The responding current for detecting the hydrogen peroxide is used to measure the amount of hydrogen peroxide and consequently determine the concentration of the glucose. Therefore, the detecting technique for the hydrogen peroxide is an important research subject. Because the sensitivity of carbon paste electrodes modified with the ruthenium hexacyanoferrate(Ⅱ) was better than that of the carbon paste electrodes modified with the ruthenium purple, the ruthenium hexacyanoferrate(Ⅱ) was used to modify the carbon paste in this research. As the ruthenium hexacyanoferrate(Ⅱ) possesses the excellent catalytic characteristic and the graphite carbon powder possesses the high conductivity, a study was conducted to use the ruthenium hexacyanoferrate(Ⅱ) to modify the carbon paste electrode and the responding current for detecting the hydrogen peroxide could then be elevated. At 30℃, -200 mV operating potential, 600 rpm stirring rate and in 0.05 M PBS buffer solution(pH=7.4), when the carbon paste electrode was modified with the ruthenium hexacyanoferrate(Ⅱ)[ruthenium hexacyanoferrate(Ⅱ)：graphite carbon powder=3：7 ( weight ratio)], the detection limit was 0.02 mM H2O2, the linear range was 0.02~2.7 mM H2O2, R2=0.9996, and the sensitivity was 661.8 µA/cm2．mM H2O2. The sensitivity for the unmodified carbon paste electrodes was 31.3 µA/cm2．mM H2O2. The results showed that the carbon paste electrode modified with the ruthenium hexacyanoferrate(Ⅱ) possessed the excellent sensitivity for detecting the hydrogen peroxide. The most suitable mixing ratio of ruthenium hexacyanoferrate(Ⅱ) to graphite carbon powder obtained from the above mentioned results could be further applied to fabricate the glucose biosensor by using the glucose oxidase which was immobilized by the Nafion on the surface of electrode.The detection limit was 0.02 mM C6H12O6 , the linear range was 0.02~2.56 mM C6H12O6 (R2=0.999), and the sensitivity was 4.11 µA/cm2．mM C6H12O6. For the hydrogen peroxide screen printed planar sensor, the detection limit was 0.02 mM H2O2, the linear range was 0.02~0.96 mM H2O2 (R2=0.917), and the sensitivity was 28.77 µA/cm2．mM H2O2. For the glucose screen printed planar sensor, the detection limit was 0.02 mM C6H12O6 , the linear range was 0.02~2.24 mM C6H12O6 (R2=0.9983), and the sensitivity was 12.19 µA/cm2．mM C6H12O6.

In 2D cell cultures uptake/release of various metabolic analytes such as glucose, lactate or metabolic by-products like hydrogen peroxide from/to the extracellular environment results in concentration gradients. The magnitude, direction, and time scales of these gradients carries information that is essential for internal cellular processes and/or for communication with neighboring cells. This PhD research work focusses on the design, fabrication and characterization of electrochemical microelectrode arrays (MEAs) optimized to be positioned in commonly used 2D cell culture setups. Importantly, by simultaneously measuring accurate concentration transients and associated gradients/uxes near the cell surface (surface concentration) the capability of the device to quantify kinetic rates and distinguish mechanisms involved in various cellular processes is demonstrated. An in-situ transient calibration technique suitable for amperometric MEAs is developed and the technique is validated by quantitatively measuring dynamic concentration profiles with varying spatial (100-800 µm) and time (s to hrs.) scales set up from an electrically controlled diffusion reaction system. With the proposed MEA design and technique three physiological applications are demonstrated. Firstly, the position able 1D MEA was employed real time to quantitatively measure the hydrogen peroxide scavenging rates from astrocyte vs glioblastoma cell cultures. With the ability to extract to dynamic surface concentration and fluxes, the cell lines were shown to have hydrogen peroxide uptake rates dependent on local surface concentrations. Moreover, the cancerous glioblastoma cells demonstrated an upregulated linear peroxide scavenging mechanism as compared to astrocytes. For the next phase, spatial scales of 1D MEA device along the size and functionalization scheme of the electrodes in the MEA was further modified to selectively sense glucose and lactate to enable extracellular metabolic profiling of cancer vs normal cell lines. Secondly, measurement of glucose concentration profiles demonstrated an increased glucose uptake rate in glioblastoma as compared to astrocytes. Additionally, sigmoidal (allosteric) vs Michaelis - Menten glucose uptake kinetics was observed in glioblastoma vs astrocytes. Moreover, the presence of a glucose sensing mechanism was observed in glioblastoma cells due to the dependence of the glucose uptake rate on initial exposed concentration rather than surface concentration. Finally, simultaneous multi-analyte (glucose and lactate) gradient measurements were performed on genetically modified mouse pancreatic cancer cell lines. While glucose uptake rate was shown to increase with increasing extracellular glucose concentration for one of the cell lines, the lactate release rate was observed to be independent of the initial extracellular glucose dose.