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Molecular recognition is an important tool for developing tunable controlled release systems and fabricating biosensors with increased selectivity and sensitivity. The development of polymer-based materials that exploit molecular recognition events such as host-guest complexation, enzyme-substrate and enzyme-inhibitor interactions and nucleic acid hybridization was pursued in this thesis. Using polymers as an anchor for molecular recognition can enhance the affinity, selectivity, and the capacity for immobilization of recognition units, enabling the practical use of affinity-based systems in real applications. To introduce the potential for immobilization while preserving or enhancing the affinity of small molecule recognition units, the affinity of derivatized cyclodextrins for the hydrophobic drug, dexamethasone, was investigated. Cyclodextrins (CDs) are molecules that possess a hydrophilic exterior and a hydrophobic cavity capable of accommodating a wide range of small molecule guests. Analysis of the solubilization capacities, thermodynamic parameters and aggregative potentials of carboxymethyl and hydrazide derivatives of CDs established the dextran-conjugated βCD derivative as an ideal carrier of hydrophobic drugs and the hydrazide βCD derivative as an optimal solubilizer of lipophilic pharmaceuticals, both alone and when incorporated in a polymer-based drug delivery vehicle. To enable non-covalent immobilization and stabilization of biomacromolecular recognition units, a printed layer hydrogel was investigated as a selective diffusion barrier for analyte sensing and enzyme inhibitor recognition. A printable hydrogel platform was developed from an established injectable system composed of aldehyde- and hydrazide-functionalized poly(oligoethylene glycol methacrylate) polymers. The printed layer hydrogel effectively immobilized a wide range of enzymes and protected enzyme activity against time-dependent and protease-induced denaturation, while facilitating the diffusion of small molecules. Furthermore, to demonstrate the potential of the printed film hydrogel immobilization layer to enhance the selectivity of the target, the printable hydrogel platform was used to develop a microarray-based assay for the screening of inhibitors of the model enzyme, β-lactamase. The assay was able to accurately quantify dose-response relationships of a series of established inhibitors, while reducing the required reagent volumes in traditional drug screening campaigns by 95%. Most significantly, the assay demonstrated an ability to discriminate true inhibitors of β-lactamase from a class of non-specific inhibitors called promiscuous aggregating inhibitors. Finally, to enable non-covalent immobilization of DNA recognition units, the printable hydrogel-based microarray was tested for its ability to immobilize DNA recognition sites and promote the detection of DNA hybridization events. A long, concatameric DNA molecule was generated through rolling circle amplification and was used as a sensing material for the detection of a small, fluorophore labeled oligonucleotide. The printable hydrogel was able to effectively entrap the rolling circle amplification product. Properties of the printable hydrogel were investigated for their ability to support the detection of DNA hybridization events. / Thesis / Doctor of Philosophy (PhD) / This thesis describes the development of polymer-based materials that exploit molecular recognition events for drug delivery and biosensing applications. First, cyclodextrins (CDs) are molecules that are capable of binding a wide range of small molecules. A comprehensive analysis of the complexation properties of CD derivatives revealed critical insight regarding their application in polymer-based drug delivery vehicles. Second, a printable hydrogel platform was developed to support the immobilization and activity of biomolecules and establish a biosensing interface that facilitates the diffusion of small molecules but not molecular aggregates. A microarray-based assay was developed by employing the printed hydrogel interface for the screening of inhibitors of the model enzyme, β-lactamase, and the detection of DNA hybridization events.

Advances in our ability to detect biological targets relevant to human health have come from the engineering of biological molecules into assemblies capable of performing target-induced signal generation. Such assemblies, known as biosensors, are composed of a molecular recognition element (MRE) and a signal generating transduction element. One MRE class that has received great attention in recent years is functional nucleic acids, which include DNA aptamers and DNAzymes. Since 1990, a large number of functional nucleic acids have been reported. However, broad commercial use of functional nucleic acids in applications that benefit human health is sparse. The goal of this thesis is to expand the usefulness of functional nucleic acids. The thesis is made of four projects. In the first project I developed a simple colorimetric biosensor for the detection of a toxic metal ion using a reported RNA-cleaving DNAzyme coupled with urease as the signal reporter. This is followed by a project where I developed a highly effective method for the synthesis and purification of the DNA-urease conjugate needed for the biosensor. I then turned my attention to the search for high-affinity DNA aptamers that bind VEGF-165, an important human protein found to be relevant in the progression of cancers. Given that VEGF-165 is a homodimeric protein, in my third project I looked into the suitability of reported DNA aptamers for this protein for the creation of dimeric aptamers with higher binding affinity. I examined multiple factors that may affect the successful engineering of dimeric aptamers and determined that none of the existing aptamers are compatible for creating a productive dimeric aptamer. With this finding, I made an effort to create our own aptamers for this protein target. I was able to isolate a new aptamer that appears to be an excellent candidate for creating a higher affinity DNA aptamer. Overall, my work adds to our increasing appreciation of the functional capability demonstrated by single-stranded DNA molecules. More importantly, I hope the methods I have developed and new functional DNA molecules I have generated in this thesis will continue to drive the development of the functional nucleic acid field and contribute to the health research community’s efforts to increase human longevity. / Thesis / Doctor of Philosophy (PhD)

Quorum sensing enables bacteria to communicate with bacteria of the same or different species, and to modulate their behavior in a cell-density dependent manner. Communication occurs by means of small quorum sensing signaling molecules (QSMs) whose concentration is proportional to the population size. When a QSM threshold concentration is reached, certain genes are expressed, thus allowing control of several processes, such as, virulence factor production, antibiotic production, and biofilm formation. Not only many pathogenic bacteria are known to produce QSMs, but also QSMs have been identified in some bacteria-related disorders. Therefore, quantitative detection of QSMs present in clinical samples may be a useful tool in the investigation and monitoring of bacteria-related diseases, thus prompting the use of QSMs as biomarkers of disease. Herein, we have developed and utilized whole-cell biosensing systems and protein based biosensing systems to detect QSMs in clinical samples, such as, saliva, stool, and bowel secretions. Additionally, since bacteria are responsible for food spoilage, we employed the developed biosensing systems to detect QSMs in food samples and demonstrated their applicability for early identification of food contamination. Furthermore, we have utilized these biosensing systems to screen antibacterial compounds employed for food preservation, namely, generally regarded as safe (GRAS) compounds, for their effect on quorum sensing.

The purpose of this study is to demonstrate the ability of surface enhanced Raman scattering (SERS) active particles to enable multiplexed immunoassays in a lateral flow format for point of care (POC) testing. The SERS particles used for this study are chemically active glass coated gold particles, containing tracer molecules which in principle can be chosen to provide Raman Spectra with unique features allowing multiple tracers to be simultaneously measured and distinguished without interference between each other. Lateral flow immunoassay technology is the important part of this study and can be conveniently packaged for the use of other than highly skilled technicians outside of the laboratory. A well-known (single channel - simplex) device for the pregnancy test is a typical example of the lateral flow assay. Similar formats have been/are being developed by others for a range of POC applications – but most diagnostic applications require simultaneous determination of a range of biomarkers and multiplexed assays are difficult to achieve without significant interference between the individual assays. This is where SERS particles may provide some advantages over existing techniques. Cardiac markers are the growing market for point of care technology therefore biomarkers of cardiac injury (Troponin, myoglobin and CRP) have been chosen as a model. The object of the study is to establish the proof of concept multiplexing assay using these chosen biomarkers. Thus, initially all different particles were characterised in single and mixture form. Also development of conjugate chemistry between antibodies for each analyte that have been purchased from commercial sources and SERS particles were analysed using different conditions like buffer, pH and antibody loading concentration to get the optimum intensity. The selected SERS particles and their conjugates were tested for size, aggregation and immune quality using a range of techniques: ultraviolet-visible (UV/Vis) absorption spectroscopy, dynamic light scattering (DLS) and lateral flow assay. These characterisations methodologies gave the understanding of optimum conditions of the each conjugates and individual’s behaviour in mixture conditions as well. After the characterisation all conjugates were tested singularly on the lateral flow assay using buffers and serum. The results of this single analyte immunoassay explained the individual’s bioactivity on the lateral flow strip. Further in study, multiplex assay have been demonstrated in serum. These outcomes have described each candidate characteristic in a mixture form on the lateral flow strip. In order to get the optimum Raman intensity from multiplex assay, the detection and capture antibodies loading concentrations were tuned in the assay. Later on different combinations (high, medium and low concentrations) of all three analytes were analysed and has found some interferences in multiplex assay. To investigate these issues various aspect were considered. First of all, different possibilities of non-specific interactions between the co-analytes and antibodies were tested. In addition, steric hindrance and optical interference investigations were performed via several assays and analysis using Scanning electron microscopy. The outcomes have confirmed related optical interferences. Therefore other assay (wound biomarkers) established to eliminate the interferences. In summary, the works reported here have built and test the equipment and necessary reagents for individual assays before moving on the more complicated task. In addition, the entire study has given a deep knowledge of multiplex assay on a single test line including the investigation of the issues for selected cardiac biomarkers and their applications in the future.

This dissertation discusses the development of inexpensive, easy-to-use, and field-deployable diagnostic techniques and devices for the early detection of various pathogens, commonly found in clinical samples and contaminated food and water. Infectious diseases account for about 90% of world health problems, killing approximately 14 million people annually, the majority of which reside in developing countries. In 2012, the World Health Organization (WHO) published data on the top 10 causes of death across the globe. Although communicable disease is a prevalent cause of fatality, both low-income and high-income countries, pathogen species and transmission are very different. Nearly 60% of deaths in developing countries are caused by food, water, air or blood-borne pathogens. The most prevalent illnesses are diarrheal disease, malaria, and HIV/AIDS. By contrast, the leading causes of death in developed countries (heart disease, cancer, and stroke) are not communicable and are often preventable. However, there is an increasing need for the development of rapid and accurate methods for pathogen identification in clinical samples, due to the growing prevalence of antibiotic-resistant strains. Incorrect, or unneeded antibiotic therapies result in the evolution of extremely aggressive nosocomial (hospital-acquired) infections, such as methicillin- (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA). The implementation of rapid, easy to use and cost-effective diagnostics will reduce the frequency of pathogen-related deaths in underdeveloped countries, and improve targeted antibiotic treatment in hospital settings, thus decreasing the potential development of more treatment-resistant "super bugs". This research includes novel techniques utilizing two major sensing modalities: serological (i.e. immunological), and nucleic acid amplification testing (NAATs). We first developed a highly sensitive (limit-of-detection = 100 CFU mL-1) particle immunoassay that takes advantage of elastic and inelastic light scatter phenomena, for optical detection of target antigens. This assay is performed upon a unique nanofibrous substrate that promotes multiplexing on a user-friendly platform. We then developed a novel technique, termed emulsion loop-mediated isothermal amplification (eLAMP), in which the target amplicon is detected in real-time, again utilizing light scattering detection and quantification. Both techniques require no sample pre-treatments, and can be combined with smartphone imaging for detection of targets in under 15 minutes. These methods have the potential to improve the speed and sensitivity of early pathogenic identification, thus leading to a reduction in preventative deaths and a decrease in global economic costs associated with infectious disease in clinical and other settings.

近年來感測器、集成電路及無線通信的科技迅速發展，促使IEEE802.15工作小組6（TG6）致力硏究一個新的無線通信標準─人體區域網路（BAN）。這個新標準特別考量在人體上、人體內或人體周邊的應用。雖然BAN至今還未達成最後定案，不同類型的應用方案已被廣泛提出。這些方案可分為醫療應用（例如：生命徵象感測和植入式治療）及非醫療應用（例如：消費性電子、個人娛樂和遙遠控制）。無線感測節點〈WSN）的基本要求包括輕巧、廉價及低耗電量。因此，本論文提出了一個符合以上要求的注入式鎖態發射機。此外，我們設計了三個發射機的內部模組。由於BAN的物理層例如調變方式和頻譜配置還未完全製訂，本文的電路設計將基於IEEE802.15 TG6的初步建議。 / 第一個模組是一個利用同相位雙路輸入及電流再使用技術的次毫瓦、第一次諧波LC注入式鎖態振盪器〈ILO）。該振盪器操作範圍在醫療植入式通訊服務〈MICS）頻段，並已採用了0.13-μm CMOS工藝實現而僅佔有200 m x 380 m芯片面積。實驗結果表明，在輸入動力0 dBm時，其鎖定範圍可達800 MHz (150 950 MHz) 。最重要的是，該ILO擁有-30 dBm的高輸入靈敏度，同時在1-V供電下只消耗660 A靜態電流。超低的靜態電流使WSN能從人體收集能量而變得完全自主。 / 第二個模組是一個低功耗MICS非整數型頻率合成器，其目的在於選擇信道。雖然整數鎖相環由於其低複雜性而被廣泛使用，對MICS頻段而言並不是一項良好方案。主要原因在於其信道寬只有300 kHz，速度、頻率解析度和相位雜訊變得很難平衡。為此，我們採用0.13-μm CMOS製程設計了一個4階第二型和差積分〈Σ-）調變器分數鎖相環。為了抑制混附單頻信號，二階單迴路數字Σ-調變器加入了抖動。仿真結果顯示該頻率合成器能在15 s內鎖定，同時在1.5-V供電下只消耗4 mW功耗。 / 第三個模組是一個高效能、完全集成的E類功率放大器〈PA）。該PA採用了自給偏壓反相器作為前置放大器，操作範圍在MICS頻段及工業、科學和醫學〈ISM）頻段。在0.18-m CMOS工藝下實現的該PA佔有0.9 mm x 0.7 mm芯片面積。實驗結果表明，在1.2-V供電下及操作頻率是433 MHz時，該PA的漏極效率及輸出功率分別可達40.2 %和14.7 dBm。當操作頻率從380 MHz 到460 MHz，該PA仍能保侍最少34.7 %的漏極效率。此設計適用於低數據傳輸率、固定振幅調變，例如：QPSK、OQPSK等。 / Recent technological advances in sensors, integrated circuits and wireless communication enable miniature devices located on, in or around the human body to form a new wireless communication standard called wireless Body Area Network (BAN). Although BAN is still being investigated by the IEEE 802.15 Task Group 6 (TG6), a vast variety of applications has been proposed which can be categorized into medical applications (e.g. vital signs monitoring and implantable therapeutic treatment) and non-medical applications (e.g. consumer electronics and remote control). The basic requirements of each Wireless Sensor Node (WSN) include light weight, small form-factor, low cost and low power consumption. This thesis proposes an injection-locked transmitter which is a potential candidate to minimize the power consumption of the RF transmitter in WSNs. Three circuit blocks in the proposed injection-locked transmitter are designed and implemented. Since the physical layer of BAN, such as modulation scheme and frequency allocation, has still not been finalized yet, the prototypes in this thesis are designed based on the preliminary suggestions made by the IEEE 802.15 TG6. / The first circuit block is a sub-mW, current-reused first-harmonic LC injection-locked oscillator (ILO) using in-phase dual-input injection technique, operating in the Medical Implantable Communications Service (MICS) band from 402MHz to 405 MHz for medical implants. It has been fabricated in a standard 0.13-m CMOS technology; occupying 200 m x 380 m. Measurement results show that the proposed ILO features a wide locking range of 800 MHz (150-950 MHz) at input power of 0 dBm. More importantly, it has a high input sensitivity of -30 dBm to lock the 3-MHz bandwidth of the MICS band, while consuming only 660 W at 1-V supply. This ultra-low power consumption enables autonomous WSNs by energy harvested from the human body. / The second circuit block is a low power MICS fractional-N frequency synthesizer for channel selection. Although integer-N phase-locked loop (PLL) is widely used due to its low circuit complexity, it is not considered as a good solution for MICS band where the channel spacing is just 300 kHz, due to the severe trade-off between speed, frequency resolution and phase noise performance. To solve this issue, a 4th-order type-II Σ- fractional-N PLL is designed using a standard 0.18-m CMOS technology. A 2nd-order single-loop digital Σ- modulator with dither is designed to eliminate the spurious tones. Simulation results verify that the synthesizer achieves 15 s locking time and consumes 4 mW at a power supply of 1.5 V. / Finally, a power-efficient fully-integrated class-E power amplifier with a self-biased inverter used as a preamplifier stage has been implemented in a standard 0.18-m CMOS process, with 0.9 mm x 0.7 mm active area. It operates in both MICS band for implantable devices and Industrial, Scientific and Medical (ISM) band for wearable devices. Experimental results shows that it achieves 40.2 % drain efficiency while output power is 14.7 dBm at 433 MHz under 1.2-V supply. Moreover, the drain efficiency maintains at least 34.7 % over the frequency range from 380 MHz to 460 MHz. This design is suitable for low data-rate, constant envelope modulation, such as QPSK, OQPSK, etc. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Kwan Wai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract of thesis entitled: --- p.I / 摘要 --- p.IV / Contents --- p.VI / List of Figures --- p.XI / List of Tables --- p.XVII / Acknowledgement --- p.XVIII / Chapter CHAPTER 1. --- Introduction --- p.1 / Chapter 1.1 --- Motivation for body area network (BAN) --- p.1 / Chapter 1.2 --- Standardization of BAN and its positioning between different communication technologies --- p.3 / Chapter 1.3 --- Classification of BAN and its potential applications --- p.5 / Chapter 1.4 --- Requirements and challenges of BAN --- p.7 / Chapter 1.5 --- Research objectives and organization of this dissertation --- p.9 / References --- p.11 / Chapter CHAPTER 2. --- Background information of biomedical transceivers --- p.12 / Chapter 2.1 --- MICS band --- p.12 / Chapter 2.1.1 --- Frequency allocation --- p.12 / Chapter 2.1.2 --- Output power --- p.13 / Chapter 2.1.3 --- Transmit spectral mask --- p.14 / Chapter 2.1.4 --- Transmit center frequency tolerance --- p.14 / Chapter 2.1.5 --- Channel model --- p.15 / Chapter 2.1.6 --- Link budget --- p.17 / Chapter 2.2 --- Fundamental figure of merits for transceivers --- p.18 / Chapter 2.2.1 --- Noise figure, noise floor and receiver sensitivity --- p.18 / Chapter 2.2.2 --- Transmitter energy efficiency --- p.19 / References --- p.20 / Chapter CHAPTER 3. --- Review of transmitter architectures --- p.21 / Chapter 3.1 --- Overview --- p.21 / Chapter 3.2 --- Architectures --- p.22 / Chapter 3.2.1 --- Quadrature --- p.22 / Chapter 3.2.2 --- Polar --- p.23 / Chapter 3.2.3 --- PLL-based --- p.24 / Chapter 3.2.4 --- Injection-locked --- p.26 / Chapter 3.3 --- Radio architecture selection for biomedical systems in BAN --- p.27 / Chapter 3.3.1 --- Data-rate --- p.27 / Chapter 3.3.2 --- Modulation scheme --- p.28 / Chapter 3.3.3 --- Proposed transmitter architecture --- p.28 / References --- p.31 / Chapter CHAPTER 4. --- Design of sub-mW injection-locked oscillator --- p.33 / Chapter 4.1 --- Introduction --- p.34 / Chapter 4.2 --- Circuit design and analysis --- p.34 / Chapter 4.3 --- Experimental results --- p.47 / Chapter 4.4 --- Summary --- p.55 / References --- p.56 / Chapter CHAPTER 5. --- Design of low-power fractional-N frequency synthesizer --- p.58 / Chapter 5.1 --- Synthesizer architectures --- p.59 / Chapter 5.2 --- PLL design fundamentals --- p.63 / Chapter 5.2.1 --- Stability --- p.63 / Chapter 5.2.2 --- Phase noise --- p.65 / Chapter 5.3 --- Proposed architecture --- p.67 / Chapter 5.4 --- System design --- p.68 / Chapter 5.4.1 --- Stability --- p.68 / Chapter 5.4.2 --- Phase noise --- p.73 / Chapter 5.5 --- Σ modulation in fractional-N synthesis --- p.75 / Chapter 5.5.1 --- Basic operating principles --- p.76 / Chapter 5.5.2 --- An accumulator as a first-order Σ- modulator --- p.78 / Chapter 5.5.3 --- Noise analysis --- p.80 / Chapter 5.5.4 --- Architectures --- p.84 / Chapter 5.5.5 --- Design and modeling --- p.87 / Chapter 5.5.6 --- Digital circuit implementation --- p.99 / Chapter 5.5.7 --- Measurement results --- p.104 / Chapter 5.6 --- Time domain behavioral modeling --- p.104 / Chapter 5.7 --- Design of building blocks --- p.106 / Chapter 5.7.1 --- VCO --- p.107 / Chapter 5.7.1.1 --- Principles --- p.107 / Chapter 5.7.1.2 --- Circuit design --- p.111 / Chapter 5.7.2 --- PFD --- p.131 / Chapter 5.7.2.1 --- Principles --- p.131 / Chapter 5.7.2.2 --- Circuit design --- p.133 / Chapter 5.7.3 --- CP --- p.136 / Chapter 5.7.3.1 --- Principles --- p.136 / Chapter 5.7.3.2 --- Circuit design --- p.137 / Chapter 5.7.4 --- Frequency divider --- p.138 / Chapter 5.7.4.1 --- Principles --- p.138 / Chapter 5.7.4.2 --- Circuit design --- p.145 / Chapter 5.7.5 --- Loop filter --- p.148 / Chapter 5.8 --- Layout issues --- p.149 / Chapter 5.9 --- Overall simulation results --- p.150 / Chapter 5.1 --- Summary --- p.152 / References --- p.153 / Chapter CHAPTER 6. --- Design of high-efficient power amplifier --- p.154 / Chapter 6.1 --- Classification of PAs --- p.154 / Chapter 6.2 --- Circuit design considerations --- p.158 / Chapter 6.3 --- Experimental results --- p.160 / Chapter 6.4 --- Summary --- p.164 / References --- p.166 / Chapter CHAPTER 7. --- Conclusions and future work --- p.167 / Chapter 7.1 --- Conclusions --- p.167 / Chapter 7.2 --- Future work --- p.168 / References --- p.171

La maladie d’Alzheimer est une pathologie neurodégénérative qui amène à une perte progressive de la mémoire et cause des changements comportementaux. Selon plusieurs théories, le développement de cette maladie est associé à l’accumulation du peptide amyloïde beta et de la protéine tau dans des zones précises du cerveau humain. A l’heure actuelle, les approches thérapeutiques testées sont fondées sur l’hypothèse de la cascade amyloïde, mais les résultats n’ont pas été jugés suffisamment efficaces. Pour augmenter les chances de succès des traitements thérapeutiques existants, de meilleures techniques pour un dépistage précoce de l’Alzheimer semblent nécessaires. De ce fait, dans cette thèse, des stratégies innovantes pour l’analyse d’un des biomarqueurs de la maladie d’Alzheimer sont proposées. En particulier le projet porte sur l’analyse de la protéine tau avec des biocapteurs basés sur la Résonance de Plasmons de Surface (SPR). L’augmentation du niveau de ce biomarqueur dans le Liquide Céphalo-Rachidien (LCR) est déjà indicateur d’un processus de neurodégénérescence. De plus, si la mesure de la protéine tau est combinée à celle d’autres biomarqueurs de la pathologie (i.e. : amyloïde beta), les possibilités de dépistage sont fortement augmentées. Les travaux ont portés sur deux aspects : initialement l’interaction antigène-anticorps a été exploitée pour développer un immunocapteur pour la protéine tau. En utilisant cette technologie, nous avons pu caractériser les paramètres analytiques de l’essai direct (avec un seul anticorps) et ceux de l’essai sandwich (avec deux anticorps complémentaires). Dès ces premières approches, nous avons remarqué le besoin d’augmenter la sensibilité de la méthode SPR développée. En effet la limite de détection pour l’essai sandwich était de l’ordre du nM, alors que les niveaux de tau dans le LCR sont de l’ordre du pM. L’utilisation de nanotechnologies, en particulier des nanotubes de carbone, a permis d’atteindre des niveaux proches du pM, avec de bonnes performances en terme de répétabilité de l’essai.Une approche alternative a été conçue dans la deuxième partie du projet. Elle était consacrée à la sélection d’un aptamère pour la protéine tau, afin d’exploiter les avantages de cette classe de récepteurs par rapport aux anticorps. Pour accomplir cet objectif, deux stratégies de sélection ont été mises en place. Premièrement la sélection traditionnelle (SELEX, Systematic Evolution of Ligands by EXponential enrichment) a été appliquée en utilisant l’Electrophorèse Capillaire (EC) comme moyen de séparation. Bien que de nombreuses conditions aient été modifiées, avec le SELEX traditionnel nous n’avons pas observé une évolution significative de l’affinité entre les séquences d’ADN et la protéine tau. Dans la deuxième approche nous avons utilisé la même méthode de séparation pour mener la sélection à travers l’EC-Non-SELEX. En utilisant cette méthode, où les étapes de PCR étaient réduites, une évolution positive a été observée après seulement trois rounds. En effet cinq séquences parmi celles issues du dernier round ont montré une affinité supérieure pour la cible par rapport à la banque. Néanmoins le nombre de séquences analysées à la fois par SPR et par anisotropie de fluorescence reste extrêmement limité par rapport au pool initial. Même si ceci semble être une limite, ce travail est le premier où les aptamères sont appliqués à l’analyse de la protéine tau. Le potentiel de cette classe de récepteurs reste en grande partie inexploré, ce qui laisse entrevoir des améliorations possibles de l’affinité grâce à de meilleurs processus de sélection et au développement de nouveaux outils bioinformatiques.En conclusion la SPR grâce à ses caractéristiques jouera un rôle fondamental dans les prochaines années pour l’analyse des biomarqueurs et pour le screening de nouvelles molécules, qui seront l’objet de futurs essais cliniques pour limiter l’agrégation de la protéine tau. / Alzheimer’s disease (AD) is a widespread pathogenic condition causing memory and behavior impairment mostly in elderlies because of the accumulation of amyloid beta peptide and tau protein in human brain. Current therapeutic approaches, based on the amyloid hypothesis, are unable to arrest the progression of the disease, hence early diagnosis is crucial for an effective intervention. Based on the updated criteria for AD probable diagnosis, and considering the limits associated with the actual analytical techniques, my work in this thesis was dedicated to develop novel strategies for AD diagnosis. The whole project focused on the analysis of tau protein by Surface Plasmon Resonance (SPR) biosensing. Such protein is well known for being relevant as neurodegenerative marker. In particular if the measurement of tau is associated with that of the amyloid beta peptide and that of the phosphorylated tau, the clinical specificity of this protein become significant to detect Alzheimer. Two aspects were studied; first of all an immunosensor was developed taking advantage of the well-established antigen-antibody interaction. After characterization of the analytical parameters of the direct assay (with primary antibody), a sandwich assay (using two monoclonal antibodies mapping on different analyte i.e. protein tau epitopes) was developed, allowing very low sensitivity to be obtained in artificial Cerebrospinal Fluid (aCSF). In particular to enhance the analytical signal Carbon Nano Tubes (CNTs) were used. Secondly, the research was focused on the selection of aptamers for tau. To this aim two SELEX (Systematic Evolution of Ligands by EXponential enrichment) methods were compared, both based on Capillary Electrophoresis (CE) for partitioning step of the process. Whether with CE-SELEX (first method), no significant affinity improvement was measured, using the CE-Non-SELEX (second method) affinity of the DNA library for tau protein was consistently improved. After isolation of a limited population of aptamer candidates, five sequences were chosen to be analyzed for their affinity for the target. Fluorescence Anisotropy (FA) measurements and SPR highlight similar behavior for the selected sequences, despite the detection principles of these techniques are significantly different. In conclusion the work highlight versatility of SPR technology used both for quantitative analysis and for new selected aptamers characterization in terms of affinity for the analyte tau. The above mentioned versatility is of great interest in a field such AD, which is rapidly expanding. Lowering the total tau levels has been recently identified as a new goal for therapy. Therefore many drug candidates are likely going to be tested in the near future. SPR technology is already widely used in pharmaceutical industry to investigate novel molecules, since it gives access to a large panel of information. In this panorama aptamer technology may improve the overall quality of the analytical data, allowing better comparison among drug candidates. With respect of these receptors, the thesis opened the door to new studies for DNA aptamers to recognize tau, with considerable advantages in term of the receptor stability. Moreover the whole potential of DNA aptamers selected in this work still remain to be explored. New selection methodologies, combined with fast progression of bioinformatics tools might give rise to affinity improvement, which will lead to sensitivity improvement for tau detection in the next few years.

Surface plasmons are collective oscillations of free electrons excited at a metal-dielectric interface by incident light. They possess a broad set of interesting properties including a high degree of tunability, the generation of strong field enhancements close to the metal's surface and high sensitivity to their adjacent dielectric environment. It is possible to enhance the sensitivity of plasmonic systems by using narrow plasmon resonances. In this thesis two approaches to narrowing surface plasmon resonances have been studied: diffraction coupling of localised surface plasmon resonances in gold nanoarrays and the use of graphene-protected copper thin films. Applications of these approaches in hybrid systems have been considered for modulation, waveguiding, biosensing and field enhancements. Arrays of gold nanostripes fabricated on a gold sublayer have been used to create extremely narrow plasmon resonances using diffraction coupling of localised plasmon resonances with quality factors up to a value of $Q \sim 300$, among the highest reported in the literature. The nanostructures were designed to give the narrowest resonance at the telecommunication wavelength of 1.5 µm, allowing for this array geometry to be used in hybrid systems for proof-of-concept optoelectronic devices. The gold nanostripe array was used in a hybrid nanomechanical electro-optical modulator along with hexagonal boron nitride (hBN) and graphene. The modulator was fabricated with an air gap between the nanoarray and the hexagonal boron nitride/graphene. Applying a gate voltage across the device moves the hBN towards the nanoarray, resulting in broadband modulation effects from the ultraviolet through to the mid-infrared dependant on the motion of the hBN instead of graphene gating. The deposition of a 400 nm hafnium(IV) oxide film on top of the gold nanoarray created a structure capable of guiding modes at 1.5 µm. The hybrid air-dielectric-stripe waveguide is capable of guiding modes over a distance of 250 µm. Copper thin films have stronger plasmon resonances and higher phase sensitivity than gold thin films. Transferring a graphene sheet on the copper prevents oxidation of the copper. A feasibility study of this hybrid system has shown that phase-sensitive graphene-protected copper biosensing can detect HT-2 mycotoxin with over four orders of magnitude greater sensitivity than commercially-available gold-based surface plasmon resonance biosensing systems. In summary, two methods of attaining narrow plasmon resonances have been demonstrated and their promise in modulation, waveguiding and biosensing have been demonstrated.