Dissertations / Theses on the topic 'Enzyme-based biosensor'

This research attempted to determine the practicality of the integration of electrospun polyaniline nanofiber as the main sensing component into interdigitated gold microelectrodes to develop a biosensor platform for sensitive, selective, and label-free detection the of Cyclooxygenase-2 (COX-2) biomarker from pure and human serum samples. COX-2 is an important enzyme in pain biomarkers, inflammation and cancer cell proliferation, so it is necessary to develop a reliable biosensor that can sensitively and objectively quantify COX-2 enzyme expression for clinical diagnosis. Polyaniline nanofibers were prepared at four different diameters using electrospinning performed at four different flow rates. The performance of the electrospun polyaniline nanofiber based biosensor was evaluated in comparison with a plain control biosensor using electrochemical impedance spectroscopy. Significant improvement was observed in the sensitivity of the electrospun polyaniline nanofiber based biosensor revealing the remarkable capability of electrospun polyaniline nanofiber in robust and rapid detection of the COX-2 biomarker. This improvement was attributed to the large specific surface area of electrospun polyaniline nanofiber as well as its highly porous structure which enhances size-matched confinement, transduction and signal strength, thus increasing the sensitivity of the biosensor significantly. The fabricated nanofiber based biosensor was able to detect the target antigen with concentrations as low as 0.01pg/ml and 10fg/ml in pure and human serum samples, respectively, as well as remarkable selectivity towards Human Serum Albumin suggesting the significant contribution of this nanofiber based platform to the enhanced strength and sensitivity in COX-2 analyte detection.
Thesis (M.S.)--Wichita State University, College of Engineering, Dept. of Mechanical Engineering

Actualment hi ha una gran preocupació sobre l'ús de pesticides en l'agricultura i els seus possibles efectes secundaris. Això fa que el desenvolupament de sistemes de detecció sensibles i robustos sigui un pas important en aquesta direcció. D'altra banda, les nano-cebes de carboni (CNOs) són materials molt atractius i prometedors amb estructures definides i propietats electroquímiques notables que amb prou feines s'han estudiat en biosensors. L'objectiu general d'aquesta tesi és estudiar la interacció de diferents plaguicides amb peroxidasa i tirosinasa amb l'objectiu de desenvolupar biosensors per a la seva detecció basats en elèctrodes modificats amb CNOs. Per aconseguir aquest objectiu general, s'ha estudiat: 1) la inhibició de les activitats de peroxidasa i tirosinasa per tres dels plaguicides més utilitzats (2,4-D, 2,4,5-T i glifosat), 2) l'ús d'CNOs oxidades com a suports per a la immobilització d'enzims i un estudi de l'activitat i estabilitat dels enzims immobilitzades, 3) el desenvolupament de biosensors electroquímics per a detecció dels plaguicides abans esmentats basats en els elèctrodes modificats amb composites contenint enzims i CNOs. Aquesta tesi és, per tant, una contribució a un camp de ràpid creixement relacionat amb el desenvolupament de noves classes de nanomaterials de carboni que té com a objectiu ampliar les seves aplicacions actuals en la construcció de sistemes de detecció nous amb millors prestacions.
Actualmente existe una gran preocupación sobre el uso de pesticidas en la agricultura y sus posibles efectos secundarios. Esto hace que el desarrollo de sistemas de detección sensibles y robustos sea un paso importante en esta dirección. Por otro lado, las nano-cebollas de carbono (CNOs) son materiales muy atractivos y prometedores con estructuras definidas y propiedades electroquímicas notables que apenas se han estudiado en biosensores. El objetivo general de esta tesis es estudiar la interacción de diferentes plaguicidas con peroxidasa y tirosinasa con el objetivo de desarrollar biosensores para su detección basados ​​en electrodos modificados con CNOs. Para lograr este objetivo general, se ha estudiado: 1) la inhibición de las actividades de peroxidasa y tirosinasa por tres de los plaguicidas más utilizados (2,4-D, 2,4,5-T y glifosato), 2) el uso de CNOs oxidadas como soportes para la inmovilización de enzimas y un estudio de la actividad y estabilidad de las enzimas inmovilizadas, 3) el desarrollo de biosensores electroquímicos para detección de los plaguicidas antes citados basados ​​en los electrodos modificados con composites conteniendo enzimas y CNOs. Esta tesis es, por lo tanto, una contribución a un campo de rápido crecimiento relacionado con el desarrollo de nuevas clases de nanomateriales de carbono que tiene como objetivo ampliar sus aplicaciones actuales en la construcción de sistemas de detección novedosos con mejores prestaciones.
There is currently a strong concern on the use of pesticides in agriculture and their possible side effects. This makes the development of sensitive and robust detection systems an important step in this direction. On the other hand, carbon nano-onions are very attractive and promising materials with defined structures and remarkable electrochemical properties that have been scarcely studied in biosensing. The overall objective of this thesis is to study the interaction of different pesticides with peroxidase and tyrosinase with the aim to develop biosensors for pesticide detection based on CNO-modified electrodes. To achieve this general objective, the following aspects have been focused on: 1) the inhibition of peroxidase and tyrosinase activities by three of the most used pesticides (2,4-D, 2,4,5-T and glyphosate), 2) the use of oxidized CNOs as supports for the immobilization of enzymes and a study of the activity and stability of the immobilized enzymes, 3) the development of electrochemical biosensors for pesticide detection based on the prepared CNO-enzyme modified electrodes. This thesis is thus a contribution to a rapidly growing field related with the development of new classes of carbon nano-onion based nanomaterials that aims at expanding their current applications in the construction of novel detection systems with improved performances.

The development of enzymatic biosensors and enzymatic biofuel cells (EBFCs) has been a significant area of research for decades. Enzymatic catalysis can provide for specific, reliable sensing of target analytes as well as the continuous generation of power from physiologically present fuels. However, the broad implementation of enzyme-based devices is still limited by low operational/storage stabilities and insufficient power densities. Approaches to improving upon these limitations have focused on the optimization of enzyme activity and electron transfer kinetics at enzyme-functionalized electrodes. Currently, such optimization can be performed through enzyme structural engineering, improvement of enzyme immobilization methodologies, and fabrication of advantageous electrode materials to enhance retained enzyme activity density at the electrode surface and electron transfer rates between enzymes and an electrode. In this work, varying electrode materials were studied to produce an increased understanding on the impacts of material properties on resulting biochemical, and electrochemical performances upon enzyme immobilization and an additional method of electroactive enzyme-based optimization was developed through the use of polymer-based protein engineering (PBPE). First, graphene/single-wall carbon nanotube cogels were studied as supports for membrane- and mediator-free EBFCs. The high available specific surface area and porosity of these materials allowed the rechargeable generation of a power density within one order of magnitude of the highest performing glucose-based EBFCs to date. Second, two additional carbon nanomaterial-based electrode materials were fabricated and examined as EBFC electrodes. Graphene-coated single-wall carbon nanotube gels and gold nanoparticle/multi-wall carbon nanotube-coated polyacrylonitrile fiber paddles were utilized as electroactive enzyme supports. The performance comparison of these three materials provided an increased understanding of the impact of material properties such as pore size, specific surface area and material surface curvature on enzyme biochemical and electrochemical characteristics upon immobilization. Third, PBPE techniques were applied to develop enzyme-redox polymer conjugates as a new platform for enzymatic biosensor and EBFC optimization. Poly(N-(3-dimethyl(ferrocenyl) methylammonium bromide)propyl acrylamide) (pFcAc) was grown directly from the surface of glucose oxidase (GOX) through atom-transfer radical polymerization. Utilization of the synthesized GOX-pFcAc conjugates led to a 24-fold increase in current generation efficiency and a 4-fold increase in EBFC power density compared to native GOX. GOX-pFcAc conjugates were further examined as working catalysts in carbon paper-based enzymatic biosensors, which provided reliable and selective glucose sensitivities and allowed a systematic analysis of sources of instability in enzyme-polymer conjugate-based biosensors and EBFCs. The knowledge gained through these studies and the in-depth characterization of an additional layer of optimization capacity using PBPE could potentially enhance the progress of enzymatic biosensor and EBFC development.

Amine transaminases (ATA) catalyse the transfer of an amino group from one molecule and replaces a ketone or aldehyde with the amino group, the amino group on the amino-donor is replaced with a ketone or aldehyde. This enzyme, ATA from Chromobacterium violaceum, has previously been used to catalyse the reaction involving amphetamine, therefore, it might be possible to use this enzyme to convert amphetamine and the product absorbs in the UV spectrum and can therefore be measured spectrophotometrically. The aim of the project was to explore the possibility of using ATA in a portable biosensor for the detection of amphetamine. A literature study of commercially available portable biosensors was performed, activity of the free enzyme was tested against two substrates, methylbenzylamine (MBA) and amphetamine. Research on immobilization techniques, materials, and surface functionalization was done to chose suitable methods for immobilizing ATA. Two immobilization methods were suggested and one of the methods, ionic immobilization through His-tag towards Ni2+ on the surface, was tested for enzyme activity toward MBA. The enzyme activity of the free enzyme in solution towards MBA was comparable to previously reported enzyme activity, however, no enzyme activity towards amphetamine was observed. No activity was observed for the immobilized enzyme, but it might be due to the experimental design, more experiments need to be performed to draw conclusions.
Amintransaminaser (ATA) katalyserar överförandet av en amingrupp från en molekyl och ersätter en keton eller aldehyd med den amingruppen, amingruppen på amin-donatorn ersätts med en keton eller aldehyd. Det här enzymet, ATA från Chromobacterium violaceum (CvATA), har tidigare använts för att katalysera en reaktion som involverar amfetamin, därför skulle detta enzym kunna användas på amfetamin. Produkten av reaktionen absorberar i UV spektrumet och kan mätas med en spektrofotometer. Målet med projektet var att utforska möjligheten av att använda CvATA i en biosensor för att detektera amfetamin. En litteraturstudie på kommersiellt tillgängliga bärbara biosensorer genomfördes, aktiviteten av det fria enzymet testades mot två substrat, metylbenzylamin (MBA) och amfetamin. Information samlades om immobiliseringstekniker, material, och ytfunktionalisering gjordes för att välja ut lämpliga metoder för immobilisering av CvATA. Två immobiliseringsmetoder föreslogs och en av metoderna, immobilisering via enzymets His6-tagg och Ni2+ joner på ytan, testades för enzymaktivitet mot MBA. Enzymaktiviteten av det fria enzymet i lösning mot MBA var i samma storleksordning som tidigare rapporterad enzymaktivitet, men ingen enzymaktivitet mot amfetamin kunde observeras. Ingen aktivitet kunde observeras för det immobiliserade enzymet, men det kan vara på grund av designen på experimentet, fler experiment behöver göras för att kunna dra några fler slutsatser.

The method of enzyme adsorption on nano- and microsized zeolites, developed by us, is described. It is notable by such advantages as simple and fast performance, the absence of toxic compounds, high reproducibility and repeatability. The biosensor based on the method developed was applied for urea measurement in samples of blood serum. It was shown that the biosensor could surely distinguish healthy people from people with renal dysfunction. Good results reproducibility was proved at urea determination in real samples of blood serum (RSD = 10%). For these reasons, the biosensors based on enzyme adsorption are more suitable for standardization and production than those based on conventional methods of immobilization. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/35478

ABSTRACT BIOSENSOR BASED ON INTERPENETRATED POLYMER NETWORK OF ALGINIC ACID AND POLY (1-VINYLIMIDAZOLE) Kartal, Mü
jgan M.S., Department of Chemistry Supervisor : Prof. Dr. Levent Toppare January 2008, 63 pages A new proton conductor polymer was prepared using alginic acid (AA) and poly (1-vinylimidazole) (PVI). The polymer network was obtained by mixing AA and PVI at various stoichiometric ratios, x (molar ratio of the monomer repeat units). The AA/PVI network was characterized by elemental analysis (EA) and FT-IR spectroscopy. Potential use of this network in enzyme immobilization was studied. Enzyme entrapped polymer networks (EEPN) were produced by immobilizing invertase and tyrosinase (PPO) in the AA/PVI network. Additionally, the maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were investigated for the immobilized invertase and enzymes. Also, temperature and pH optimization, operational stability and shelf life of the polymer network were examined.

Biosensors have proven to be a powerful tool for detecting diverse targets, such as proteins, DNA, and small molecules representing disease biomarkers, toxins, drugs and their metabolites, environmental pollutants, agrichemicals, and antibiotics with high sensitivity and specificity. The major objective of the research described in this dissertation was to develop low cost, low sample volume, highly sensitive and specific AuNP-based colorimetric sensor platforms for the detection of DNA and small molecules. With this in mind, we propose an instrument-free approach in chapter three for the detection of NADH with a sensor constructed on a paper substrate, based on the target-induced inhibition of AuNP dissolution. The successful detection of this important molecule opens the door to numerous possibilities for dehydrogenase characterization, because NAD+/NADH are essential cofactors for more than 300 dehydrogenase enzymes. To further increase the sensitivity of our hybridization-based assay for DNA detection, we developed an enzyme-assisted target recycling (EATR) strategy in chapter four and have applied such an EATR-based colorimetric assay to detect single-nucleotide mismatches in a target DNA with DNA-functionalized AuNPs. This assay is based on the principle that nuclease enzymes recognize probe–target complexes, cleaving only the probe strand. This results in target release, enabling subsequent binding to and cleavage of another probe molecule. When the probe is conjugated onto AuNPs, complete cleavage from the AuNP surface produces a detectable signal in high ionic strength environments as the nanoparticles undergo aggregation. With such enzyme-assisted amplification, target detection can occur with a very low nM detection limit within 15 minutes. The extent of DNA loading on the AuNP surface plays an important role in the efficiency of DNA hybridization and aptamer-target assembly. Many studies have shown that high surface-coverage is associated with steric hindrance, electrostatic repulsive interactions and elevated surface salt concentration, whereas low surface-coverage can result in nonspecific binding of oligonucleotides to the particle surface. In chapter five, we investigated DNA surface coverage effects, and apply this optimization in conjunction with a highly-specific aptamer to develop a sensitive colorimetric sensor for rapid cocaine detection based on the inhibition of nuclease enzyme activity.

>Magister Scientiae - MSc
Glucose oxidase (GOx) and horseradish peroxidase (HRP) are important enzymes for the development of amperometric enzyme linked immunosensors. The selectivity of each enzyme towards its analyte deepens its importance in determining the sensitivity of the resultant immunosensor. In designing immunosensors that have customized transducer surfaces, the incorporation with FAD and iron based enzymes ensures that electron kinetics remains optimal for electrochemical measurement. Various different immobilization strategies are used to produce response signals directly proportional to the concentration of analyte with minimal interferences. The combination of self-assembled monolayers and supramolecular chemistry affords stability and simplicity in immunosensor design. In this work, two electrochemical strategies for the detection of human chorionic gonadotropin(hCG) is presented. This involves the modification of a gold surface with a thiolated β-cyclodextrin epichlorohydrin polymer (βCDPSH) to form a supramolecular inclusion complex with ferrocene (Fc)-functionalised carboxymethyl cellulose polymer (CMC). Cyclic voltammetry indicated that ferrocene is in close proximity to the electrode surface due to the supramolecular complex formed with βCDPSH. Furthermore, strategy (a) for the detection of hCG used α-antihCG labelled (HRP) as reporter conjugate. Strategy (b) maintained the CMC bifunctionalised with Fc and recognition antibody for hCG hormone. However, the system was functionalised with a HRP enzyme and detection is done by using GOx reporter conjugates for in situ production of hydrogen peroxide. The reduction of H2O2 was used for the amperometric detection of hCG by applying a potential of 200 mV. The sensitivity and limit of detection of both strategies were calculated from calibration plots. For strategy (a) the LOD was found to be 3.7283 ng/mL corresponding to 33.56 mIU/mL and a sensitivity of 0.0914 nA ng-1 mL-1. The corresponding values for strategy (b) are 700 pg/mL (6.3 mIU/mL) and 0.94 nA ng-1 mL-1.

Diabetic ketoacidosis (DKA) is a life-threatening condition that can appear in patients with diabetes. High ketones in the blood lead to acidity of the blood. For DKA diagnosis and management, ketones such as hydroxybutyrate (HB) can be used to quantify the severity of the disease. The fabrication of electrochemical biosensors for the detection of HB is attractive since their capability to deliver fast response, high sensitivity, good selectivity and potential for miniaturisation. In this thesis, an integrated electrode system was prepared for the detection of HB. Laser-induced graphene (LIG) with a 3D porous structure was used as the flexible platform. Poly (toluidine blue O) (PTB) was electro-deposited on LIG (PTB/LIG) under the optimised conduction (pH of 9.7 and from 0.4 to an upper cyclic potential of 0.8 V). The single PTB/LIG working electrode demonstrated excellent performance towards the detection of NADH with a linear range of 6.7 M to 3 mM using chronoamperometry, high sensitivity of detecting NADH and excellent anti-fouling ability (94 % response current retained after 1500 s). Further integration of the 3-electrode system realised the static amperometric detection of NADH over the range of 78 M to 10 mM. Based on the excellent performance of PTB/LIG to NADH sensing, hydroxybutyrate dehydrogenase was immobilised via encapsulation with chitosan and polyvinyl butyral (PVB) which was used for HB biosensing over the linear range of 0.5 M to 1 mM with NAD+ dissolved in solution. In addition, the co-immobilisation of NAD+ and HBD on PTB/LIG was conducted by optimisation of enzyme and NAD+ amount per electrode, which shows excellent reproducibility and satisfactory HB biosensing performance. Further experiments to improve the long-term stability of the enzyme electrode is expected in the future. The proposed integrated electrode system also possesses the potential to extend to a multichannel sensor array for the detection of multiple biomarkers (e.g. pH and glucose) for diagnosis and management of DKA.

Un papier bioactif est obtenu par la modification d’un papier en y immobilisant une ou plusieurs biomolécules. La recherche et le développement de papiers bioactifs est en plein essor car le papier est un substrat peu dispendieux qui est déjà d’usage très répandu à travers le monde. Bien que les papiers bioactifs n’aient pas connus de succès commercial depuis la mise en marche de bandelettes mesurant le taux de glucose dans les années cinquante, de nombreux groupes de recherche travaillent à immobiliser des biomolécules sur le papier pour obtenir un papier bioactif qui est abordable et possède une bonne durée de vie. Contrairement à la glucose oxidase, l’enzyme utilisée sur ces bandelettes, la majorité des biomolécules sont très fragiles et perdent leur activité très rapidement lorsqu’immobilisées sur des papiers. Le développement de nouveaux papiers bioactifs pouvant détecter des substances d’intérêt ou même désactiver des pathogènes dépend donc de découverte de nouvelles techniques d’immobilisation des biomolécules permettant de maintenir leur activité tout en étant applicable dans la chaîne de production actuelle des papiers fins. Le but de cette thèse est de développer une technique d’immobilisation efficace et versatile, permettant de protéger l’activité de biomolécules incorporées sur des papiers. La microencapsulation a été choisie comme technique d’immobilisation car elle permet d’enfermer de grandes quantités de biomolécules à l’intérieur d’une sphère poreuse permettant leur protection. Pour cette étude, le polymère poly(éthylènediimine) a été choisi afin de générer la paroi des microcapsules. Les enzymes laccase et glucose oxidase, dont les propriétés sont bien établies, seront utilisées comme biomolécules test. Dans un premier temps, deux procédures d’encapsulation ont été développées puis étudiées. La méthode par émulsion produit des microcapsules de plus petits diamètres que la méthode par encapsulation utilisant un encapsulateur, bien que cette dernière offre une meilleure efficacité d’encapsulation. Par la suite, l’effet de la procédure d’encapsulation sur l’activité enzymatique et la stabilité thermique des enzymes a été étudié à cause de l’importance du maintien de l’activité sur le développement d’une plateforme d’immobilisation. L’effet de la nature du polymère utilisé pour la fabrication des capsules sur la conformation de l’enzyme a été étudié pour la première fois. Finalement, l’applicabilité des microcapsules de poly(éthylèneimine) dans la confection de papiers bioactifs a été démontré par le biais de trois prototypes. Un papier réagissant au glucose a été obtenu en immobilisant des microcapsules contenant l’enzyme glucose oxidase. Un papier sensible à l’enzyme neuraminidase pour la détection de la vaginose bactérienne avec une plus grande stabilité durant l’entreposage a été fait en encapsulant les réactifs colorimétriques dans des capsules de poly(éthylèneimine). L’utilisation de microcapsules pour l’immobilisation d’anticorps a également été étudiée. Les avancées au niveau de la plateforme d’immobilisation de biomolécules par microencapsulation qui ont été réalisées lors de cette thèse permettront de mieux comprendre l’effet des réactifs impliqués dans la procédure de microencapsulation sur la stabilité, l’activité et la conformation des biomolécules. Les résultats obtenus démontrent que la plateforme d’immobilisation développée peut être appliquée pour la confection de nouveaux papiers bioactifs.
Biosensing paper attracts increasing attention due to its benefits of being simple, visible, portable and useful for detecting various contaminants, pathogens and toxins. While there has been no bioactive paper commercialized since glucose paper strips developed in the fifties, many research groups are working to immobilize biomolecules on paper to achieve a bioactive paper that is affordable and has good shelf life. The goal of this research is to develop some highly useful bioactive paper that could, for example, measure blood glucose, or immediately detect and simultaneously deactivate pathogens such as neuraminidase and E.coli. Previously, bioactive paper was produced either through physically absorbing biorecognition elements or printing bio-ink onto paper substrate. Our methodology for fabrication of bioactive paper strips is compatible with existing paper making process and includes three procedures: the fabrication of microcapsules, enzyme or antibody microencapsulation, immobilization of enzymes or antibody-entrapped microcapsules into paper pulp. The first step, in fabricating of bioactive paper strips is to produce biocompatible and inexpensive microcapsules with suitable parameters. To do so, two types of microencapsulation methods were compared; the emulsion method and the vibration nozzle method accomplished with an encapsulator. The parameters for producing optimal microcapsules with both methods were studied. Factors that affect their diameter, wall thickness, shell pore size, encapsulation efficiency and membrane compositions were also discussed. By comparison, microcapsules prepared with poly(ethyleneimine) (PEI) by the emulsion method exhibit properties that were more suitable for enzyme encapsulation and paper making process, whereas the microcapsules prepared by the vibration nozzle method were too big to be immobilized within paper pulp, and had lower encapsulation efficiency, enzymatic activity and productivity. Thus the emulsion method was chosen for subsequent experiments such as enzyme and antibody microencapsulation and bacterial vaginosis (BV) paper preparation. Microcapsules made by the emulsion method were semi-permeable in that the diffusion of substrate and product molecules were allowed freely across the membranes but the encapsulated enzymes would be retained inside. Glucose oxidase from Aspergillus niger (GOx) and laccase from Trametes versicolor (TvL) microcapsules showed high encapsulation efficiency, but the encapsulation process caused a severe decrease in the specific activities of both enzymes. Results from circular dichroism (CD) studies, fluorescence properties, enzymatic activities of free enzymes and Michaelis-Menten behavior demonstrated that the Vmax decrease for GOx was due to the restriction of diffusion across microcapsule membranes with pore size less than 5 nm. The microencapsulation process improved the thermal stability of GOx but decreased that of laccase. Bioactive papers were fabricated either by incorporating microcapsules containing different enzymes or empty microcapsules soaked in substrate and enhancer solution into the paper pulp during the sheet making process. Both the GOx and the BV paper strips underwent a color change in the presence of glucose and potassium iodide, and sialidase from Clostridium perfringens respectively. Some preliminary studies on antibody sensitized microcapsules, in which antibody was either encapsulated within the PEI microcapsules or conjugated to its membranes, were also performed. Our objective was to establish an enzyme immobilization platform based on microencapsulation techniques for paper based biosensors. Even though our current studies only focused on the microencapsulation of two enzymes, TvL and GOx, as well as the bioactive paper preparation, a similar approach can be applied to other enzymes. We believe that this immobilization method can potentially be employed for bioactive paper preparation on an industrial scale.

碩士
淡江大學
化學學系碩士班
102
Two parallel enzymatic oxygen-consumed reaction including oxidation of hypoxanthine by xanthine oxidase (EC 1.17.3.2) and oxidation of catechol by tyrosinase (EC 1.14.18.1) were utilized to constructed novel allopurinol biosensor. In this project, the concentration increase of allopurinol (inhibitor) would inhibit the activity of enzymatic oxygen-consumption by xanthine oxidase. Subsequently, tyrosinase could divvy more oxygen to produce catechol quinone, and it could be observed that more current response was recorded from electrochemical reduction of catechol quinone at 0.0V (vs. Ag/AgCl). In contrast to the determination of traditional inhibition type which the signal was inverse proportional to the concentration of inhibitor, the signal is proportional to the concentration of allopurinol. Moreover, in this biosensor, the monolayer of enzyme modifier and 0.0V detection potential were adapted to obtains fast response (t90%-10% = 2.9 second) and efficiently avoids common interference of substances that co-exist in serum. This allopurinol biosensor possesses linear range 5μM－100μM (R=0.998) that satisfy with therapeutic range (5-15mg/L), sensitivity is 8.79 nA/μM, detection limit is 1.4μM, the relative standard deviation (RSD) is 4.2%. Allopurinol was used to primary treatment of hyperuricemia and its complication. However, it potentially causes serious hypersensitivity such as Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TENS) on several patients after allopurinol treating. Therefore, the determination of allopurinol is important and necessary for clinical monitoring and quality assurance of pharmacy.

The present thesis focuses on the application of two distinct enzymes – cytochrome c nitrite reductase (ccNiR) and paraoxonase 1 (PON1) – as the key elements in the development of two electrochemical analytical devices, namely a 3rd generation miniaturized biosensor for nitrite quantification, and a 1st generation biosensor for the detection of homocysteine thiolactone (HTL). The analytical surveillance of nitrite is crucial in the management of public health and environmental safety. Additionally, it is considered an important marker of proper endothelial function and a common indicator of urinary tract infection. The endogenously produced HTL has been implied in the pathophysiology of the vascular system, making the detection and quantification of this metabolite necessary in the study and diagnosis of processes related with cardiovascular diseases. The biocompatibility of ccNiR with carbon conductive inks and low-temperature curing processes was previously demonstrated on standard pyrolytic graphite electrodes. In this work, the next step in the development of a disposable miniaturized nitrite biosensor was performed, by manually applying a carbon ink and ccNiR composite on carbon-screen printed electrodes. The analytical performance was evaluated by cyclic voltammetry, and the bioelectrodes obtained in this way showed good catalytic response towards nitrite, although with poor reproducibility. Future automated fabrication processes could improve this. Nonetheless, the successful incorporation of the catalytically competent and stable enzyme, indicate a putative application for future co-printing with the transducing electrode. Due to the need of an oxygen-free environment, two enzymatic deoxygenation systems were integrated with the transducer: one based on the well-known couple glucose oxidase/catalase, while the other was a novel system based on multicopper oxidases. Overall, the combination of these enzymes with ccNiR on carbon screen-printed electrodes resulted in miniaturized nitrite voltammetric biosensors capable of operating at low potentials, under ambient air. Additionally, for the measurement of nitrite dynamics in cerebral tissue, carbon fiber microelectrodes and ceramic-based platinum microelectrodes arrays were employed as transducers in the development of microscale (bio)sensing platforms. Preliminary data showed that exogenous nitrite real-time clearance could thus be detected in situ and in vivo. Owing to the putative relation between lower PON1 activity and the greater risk of developing diseases with an inflammatory component, a novel electrochemical protocol for PON1 status determination was developed. Additionally, the potential role of the enzyme as the bioreceptor in an HTL voltammetric biosensor was also addressed.

The rise in the prevalence of chronic illnesses in under-developed countries, such as Diabetes mellitus, increases the necessity for diagnosis and medical treatment in these areas. According to the World Health Organization (WHO), diabetes is one of the most prevalente diseases globally, with close to 400 million patients, being predicted that this number will rise in the future. Consequently, there is the necessity to create alternatives that combat the lack of accessibility to medical facilities and increase the efficiency in diagnosis and treatment. In this context, precise and accessible measurement of relevant biomarkers, for diabetes and other complications, is of extreme importance. The creation of point-f-care devices, produced from a versatile and abundant material such as paper, in combination with enzyme-free, colorimetric detection methods, paired with information systems such as smartphones, stand out as a simple, robust and low-cost alternative, which allows for quantitative results in a short period of time. Especially, the potential to create multiplex sensors, measuring various markers simultaneously, using the same biological sample, brings a significant improvement in the efficiency of these measurements. The main goal for this dissertation work is, then, to develop a multiplex biosensor, able to simultaneously measure physiological concentrations of glucose, uric acid and cholesterol. To do so, Lab-on-Paper technology was employed, to create paper-based devices, using hydrophobic wax printing to develop microfluidic channels. This device is paired with enzyme-free detection methods, based on the use of gold nanoparticles, which are manipulated to show affinity towards these biomarkers. The developed multiplex sensor results from the application of recognition assays in paper substrate, using the colorimetric properties of reactions between the target analytes and gold nanoparticles. These assays were tested with solutions of different concentrations inside each respective physiological range. These tests were submitted to digital analysis, resulting in calibration curves which allow for the extrapolation of concentrations for each of the biomarkers, in a simple, fast and economical way.

碩士
國立雲林科技大學
電子工程系
104
In this thesis, it was mentioned the enzymatic glucose biosensor was manufactured by using radio frequency sputtering system, thermal evaporation system and screen-printed technology, whose glucose sensing membrane was composed of indium gallium zinc oxide (IGZO) membrane and glucose oxidase (GOx). For enhancing sensing characteristics of enzymatic glucose biosensor, the sensing membrane was modified by graphene oxide (GO) and magnetic beads (MBs) to improve adsorption of enzyme and sensing characteristics. According to experiential results, the average sensitivity and linearity of enzymatic glucose biosensor modified by GO and MBs were 10.391 mV/mM and 0.998, respectively. To demonstrate that sensing membrane was successfully modified by GO and MBs, the electrochemical impedance spectroscopy (EIS) was used to analyze the capability of electron transfer for sensing membranes. The stability, lifetime, interference and detection limit of the enzymatic glucose biosensor modified by GO and MBs were investigated. Finally, the enzymatic glucose biosensor modified by GO and MBs was integrated with the microfluidic framework and the sensing characteristics under dynamic conditions, i.e., solution under flowing condition, were investigated. According to experiential results, under dynamic conditions, the average sensitivity and linearity of enzymatic glucose biosensor modified by GO and MBs were enhanced to 12.383 mV/mM and 0.999, respectively. Furthermore, in order to develop the real-time sensing system applied in measurement of pH value and multifunctional enzyme, the pH sensor as well as enzymatic glucose, lactate and urea biosensor modified by GO and MBs was combined with wireless sensing system to carry out the wireless sensing measurements, and this system complied with ZigBee wireless networking protocol which consisted of the XBee module, Arduino Mega 2560, readout circuit, biosensor and computer was employed to transmit the measurement signals. According to the experimental results, the average sensitivities of the pH sensor as well as enzymatic glucose, lactate and urea biosensor modified by GO and MBs were 50.059 mV/pH, 10.257 mV/mM, 55.747 mV/mM and 2.066 mV/(mg/dl), respectively.

碩士
中原大學
電子工程研究所
92
The detection and determination of urea are the important parameters and great interests in biomedical and clinical analysis applications where a reduced level in urine and an increased concentration in blood are the important indication for renal dysfunction. Although there are direct spectrophotometric methods of urea determination, enzymatic methods are more selective and much more widely used. A potentiometric solid-state urea biosensor prepared by immobilization of urease directly onto the surface of an ammonium ion-selective electrode is described in this paper. The substrate of the ammonium ion-selective electrode is the tin oxide (SnO2) / indium tin oxide (ITO) glass and the enzyme was immobilized by entrapment method on a nonactin membrane incorporated carboxylated polyvinylchloride (PVC-COOH). However, for the quantitative expression for their selectivity is desired, the article employed the selectivity coefficients approach to estimate the selectivity of urea biosensors. In this study, experimental procedures based on the Nicolsky-Eisenman equation were considered. Therefore, the level of influence of the preference of the urea biosensors on the primary ions relative to interfering ions of is determined. According to this paper, urea biosensors based on ammonium ion-selective electrodes respond rapidly and stable within the pH range from pH 6 to pH 7.5. The slope in the linear range (0.026 mM to10 mM) is about 55.56±3.15 mV/decade and the detection limit is 5 μM. Moreover, the use of ammonium ion-selective electrode has the advantage of providing solid-state urea biosensors that are easy to fabricate, good reproducibility and stability, low cost and allow miniaturization configuration.

碩士
國立成功大學
醫學工程學系
81
Most of the electrochemical reaction phenomena and its equations are relatively complicate, therefore, it is difficult to estimate the kinetic parameters exactly from some experimental processes. Recently, the digital computer had been designed in high-speed operation and many simulation methods were reported, it promoted the development in bioelectrochemical analysis. Among these researches, the digital simulation systems based on explicit finite difference method was utilized to discuss the electrochemical behaviors and showed the excellent possibilities in each cases containing homogeneous enzyme reactions. Here, we further considered the mechanism of enzyme reaction and diffusion of mass transfer to the simulation process. Now, the electrochemical characteristics of redox compound, an electron transfer mediator, and electrooxidation of NADH based on diaphorase (Dp) and ferrocenylmethylalcohol (FMA) were discussed. The simulated values of diffusion coefficients of FMA and the Michaelis constant of Dp were proved to be extremely agreed to the reported values. At present, we are making an effort on the case of enzyme- immobilized electrode. In this analytical process, the partition number $(\alpha)$ and diffusion coefficient ($D_m$) of mediator in enzyme membrane were required. For this purpose, potential step analysis followed by rotating method was applied. We believe this simulation system would be not only useful in efficiency test of enzyme electrode, but also helpful in finding out the optimal condition for enzyme membrane fabrication.