In recent years there has been an increased drive towards point of care testing (POCT), in which assays are performed at the site of the patient. This has many benefits, critically; the time for a result to be obtained will be significantly reduced, allowing for greater and more effective decision making. Many currently used bioassay methods are not affordable in resource poor areas where infectious disease is most prevalent, in order to combat this issue many research groups are attempting to miniaturise equipment for portability and make assays more affordable and therefore more accessible. With the aims of generating a new assay platform which is highly portable and affordable, the work in this thesis presents the development of several generic methods utilising nano- and micro-scale beads coated with aptamer which are then monitored interacting with target proteins with Tunable Resistive Pulse Sensing (TRPS). Aptamers are short oligonucleotide sequences which are capable of binding to a wide range of targets with high selectivity and comparable affinity to antibodies while possessing greater stability and have begun to challenge the role of antibodies. When aptamers bind a target, they often undergo a conformational change. In the assays described herein, this conformational change is key to the observed signal changes. TRPS is a pore-based system in which beads moving through a pore cause a measurable increase in resistance which can be used to derive particle size, concentration, and mobility. During the course of this thesis several template TRPS aptasensors have been developed. TRPS was successfully used to confirm the successful coating of nano- and micro-scale beads with DNA aptamers by monitoring an increase in electrophoretic mobility when the negatively charged DNA is added to the surface. Following on from this, TRPS was used to monitor the interaction of aptamer tagged beads with thrombin protein enabling thrombin detection down to 1.4 nM and the comparison of several thrombin-aptamers with results comparable to previously published SPR data. Thrombin was postulated to shield the negative DNA, resulting in a decrease in mobility, and the magnitude of this charge shielding was found to depend upon the binding mechanism of the aptamer used. This effect is not thought to be specific to our system nor to thrombin, the principles outlined here may be applied to other RPS technologies, or by interchanging of the aptamer, different proteins. In later chapters, this method is expanded to include multiplexed detection of growth factors and a significant improvement in signal. vi Following on from this, the controlled aggregation of avidin coated beads in the presence of biotinylated-BSA was explored. Factors impacting upon this assay were discussed including magnetic separation, particle size and particle concentration, and different methods of data interpretation were presented. This aggregation study identified several key parameters in the use of TRPS in aggregation assays. Using the methods outlined by the study of aggregates, a dispersion assay was then designed in which the interaction of thrombin proteins with clusters of particles brought about the release of many small particles by the disruption of double stranded DNA linkages. This dispersion assay incorporated magnetic separation to simplify the read-out and relied on measuring particle concentration rather than mobility, enabling the use of additional pressure to increase speed and ease of use. Using this method, thrombin was able to be detected down to 100 fM, a significant advancement in TRPS aptasensors.

Au cours de ce projet de thèse, l'imagerie optique a été utilisé pour étudier la biodistribution de différents types de nanoparticules pour un ciblage passif de tumeur et plusieurs aptamères sélectionnées par SELEX in vivo pour un ciblage actif, dans des modèles murins de cancer. Pour la partie ciblage passif, l'effet de l’enrobage zwitterionique sur la biodistribution de micelles a été étudié. L'imagerie planaire a montré qu’il y avait un ciblage tumoral passif à partir de 24 h post-injection. Apres 24h, les micelles ne sont pas retenues, par opposition aux micelles PEGylés. Concernant la biodistribution des nanogels, l’imagerie planaire a montré un ciblage tumoral passif à 24 h. Un ciblage passif de la tumeur a été également observé pour l'un des polymères, tandis que l'autre n'a pas été retenu. Pour la partie ciblage actif, seulement quelques séquences ont montrés un potentiel un ciblage des tumeurs. Cependant, des investigations plus approfondies sur les séquences prometteuses sont nécessaires afin de valider leur captation par le tissue tumoral.En outre, une corrélation linéaire a été observée entre l’imagerie planaire in vivo et ex vivo, illustrant l'utilité de l'imagerie optique pour fournir des informations de base sur la biodistribution et d'élimination des nanoparticules et les aptamères.En conclusion, le travail effectué au cours de cette thèse devrait permettre d'ouvrir de nouvelles perspectives pour le développement de nano-objets multimodales qui peuvent être utilisés pour des applications dans le diagnostic et même pour l'administration spécifique de médicaments à des sites de la maladie. / During this PhD project, optical imaging was used to study the biodistribution of different types of nanoparticles for passive tumor targeting and several candidate aptamers selected by in vivo SELEX for active tumor targeting, in animal models of cancer. For the part of passive tumor targeting, the effect of zwitterionic coating on the biodistribution of polydiacetylenic micelles was investigated. Planar fluorescence imaging demonstrated passive tumor targeting during 24 h but the micelles were not retained over time as opposed to PEGylated micelles. The biodistribution of two new types of nanogels and their constituent polymers was also evaluated. Planar imaging showed passive tumor targeting 24 h for the two nanogels. Surprisingly, tumor targeting was also observed for one of the polymers, while the other was not retained.For the part of active tumor targeting, only few sequences displayed potentials for active tumor targeting. However, further investigation of these promising sequences is needed in order to validate their favorable tumor uptake.Moreover, linear correlation was observed between in vivo and ex vivo planar imaging, demonstrating the utility of optical imaging to provide basic preclinical information regarding biodistribution of nanoparticles and aptamers.In conclusion, the work done during this thesis should help open new perspectives to the development of multimodal nano-objects that can be used for applications for tumor diagnosis and even drug delivery to sites of disease.

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Functional nucleic acids (FNAs), which include both DNA/RNA aptamers and DNA enzymes, have emerged as promising biological recognition elements for biosensors. These species typically require immobilization on or within a solid support, which is usually interfaced to some kind of signal transducer and readout system when use in biosensor. Our group has successfully immobilized several functional nucleic acids in the past, including fluorescence-signalling DNA enzymes, DNA aptamers and RNA aptamers by entrapping them into porous silica or organosilica materials prepared by the sol-gel method using percursors such as sodium silicate (SS), diglyceryl silane (DGS), tetrametylorthosilicate (TMOS) and trimethoxymethysilane (MTMS). While the earlier work established the ability of entrapped FNAs to retain binding and catalytic activity, only limited information was obtained on how different factors affect the performance of immobilized FNAs, and no information was obtained on the effects of aging and storage conditions on FNA performance. The initial objective of this thesis was to employ advanced fluorescence methods to better understand the nature of immobilized DNA and RNA aptamers, and in particular how entrapment in different sol-gel based materials affected FNA performance for detection of small molecule analytes. It was found that the ability of the entrapped aptamer reporters to remain fully hybridized was the most important factor in terms of signalling capability for both DNA and RNA aptamer reporters. It was also observed that more polar materials derived from SS were optimal for both types of aptamer reporters, since these allowed the entrapped aptamers to remain hydridized to their complementary strands and still retain the dynamic motion needed to undergo structure switching, while providing a minimum degree of leaching. The second objective of my research was to develop a paper-based biosensing device incorporating immobilized DNA and RNA aptamers that could be used in the fields of point-of-care diagnostics to further expand the utility of structure-switching aptamer reporters to real world application. A dual response (fluorescence / colorimetric) paper-based sensor utilized printed graphene oxide to immobilize both a RNA and a DNA aptamer in a recognition zone. Upon target addition, the aptamer desorbed and eluted to an amplification zone where rolling circle amplification was used to generate a colorimetric output. This sensor could function with clinical samples such as serum and stool, and allowed detection of key bacterial markers (ATP and glutamate dehydrogenase) at clinically relevant levels. / Thesis / Doctor of Philosophy (PhD)

An efficient delivery system and patient compliance are two of the most important factors for any drug delivery system design to be successful. The current standard, particularly to the ocular anterior segment, are topical applications including eye drops. However, due to ocular physical barriers including blinking, the varying tear film layers, and the structure of the corneal epithelium, less that 5% of drug reach the target tissue from a single eye drop dose. While most treatment regiments combat this with increased frequency of dosage and higher than needed concentrations, the need for a more efficient and controlled system has been recognized to reduce the risk of possible side effects. Contact lenses (CL) have been a widely discussed potential drug delivery device given their accepted use in the population, their ability to hold drug, as well as their placement on the ocular surface. The current work focuses on testing a novel delivery system using CLs with the incorporation of drug specific oligonucleotide chains known as aptamers on the surface of the lenses. This application of contact lenses is aimed at capitalizing on the strong affinity of aptamers to hold drug on the surface of the lenses until they are applied to the eyes. The aptamers were covalently attached to the surface via the activation of the hydroxyl groups on pHEMA as a model lens material using 1’1-carbonyldiimidazone CDI chemistry and subsequent reaction with the amine group on the 5' end of the aptamer. The presence of aptamers was confirmed using 6-carbofluorescein (6-FAM) fluorescence detection and x-ray photoelectron spectroscopy (XPS). The release of kanamycin B in comparison to regular pHEMA gels using a soaking uptake method was assessed. In this work, aptamers were confirmed through fluorescence to have been successfully reacted onto the surface, however XPS was not able to confirm a consistent reading. This may have been due to low initial amounts of aptamer or uneven distributions along the surface. The efficiency of the aptamer reaction was not tested and would need to be further investigated. The contact angle had a significant change with increased hydrophilicity at 60.7 ± 1.55° compared to 66.6 ± 0.67°, however physically it should not affect wettability. The lower aptamer amounts resulted in no significant difference during drug release. Kanamycin B was detected using liquid chromatography mass spectroscopy (LCMS) with a reverse phase method using a C18 column however quite a few errors in the methodology led to the conclusion that this method of drug release requires further investigation. It is recommended an aptamer-surface reaction efficiency be determined with the use of a much larger starting aptamer amount, as well as a follow up drug release. / Thesis / Master of Applied Science (MASc)

Aptamers are short nucleic acid ligands generated by the process of iterative selection. Nucleic acid counterparts to protein antibodies, aptamers bind their targets with relatively high affinities by assuming characteristic shapes. Highly thermostable, open to manipulations and non-immunogenic, these olignucleotides can be readily adapted to a variety of diagnostic assays and harvested for their therapeutic potential. We have particularly focused on the unique prospects that stem from their localization patterns both in vitro and in vivo. While several assays exist for protein diagnostics, many of these are limited by the amount of target they can detect. To overcome these limitations it might prove effective to couple protein detection with nucleic acid based amplification. The Proximity Ligation Assay (PLA) is an innovative technique that facilitates protein detection on a zeptomolar range by amplifying a tiny signal via the polymerase chain reaction. PLA is based on the concept that two DNA tags when co-localized adjacent to one another on a protein surface and ligated via a connector nucleotide will form a unique amplicon that can detected using real-time PCR and in turn detect the protein. We have adapted PLA to the peptide based detection of Bacillus spores as well as the RNA aptamer based detection of cancer cells. Highly sensitive and specific, nucleic acid based PLA could serve as a promising tool in diagnostics. Aptamers have also been analyzed for their localization patterns in vivo. Using two anti-prostate specific membrane antigen RNA aptamers, we have demonstrated that there is an inherent bias for some circulating oligonucleotides over others based solely on their sequence. This phenomenon has also been explored in cancer models of mice for persistence of specific aptamers over others in tumors for therapy. An in vivo “Stealth” selection scheme has also been designed and executed to hunt for stable and robust aptamer species that are naturally chosen for survival within a mouse system. Generation of such ligands could benefit several therapeutic ventures such as targeted drug delivery past complex vasculature as in the case of the blood:brain barrier. / text

>Magister Scientiae - MSc / Tuberculosis (TB) is a global health problem and rated as the second leading cause of death after HIV/AIDS. Transmission of TB from one person to the next is very rapid in crowded communities. Therefore, it is crucial to identify people who are infected as quickly as possible not only to provide treatment but also to prevent the spread of the disease. Current TB diagnostic tests such as the culture and sputum smear tests are time-consuming, while rapid tests make use of antibodies that are costly and have low sensitivity and stability. Great improvement has been observed when aptamers are used in place of antibodies in rapid diagnostic tests such as lateral flow devices (LFDs). Therefore, the current study aims to synthesize and identify aptamers against serum biomarkers for development of rapid TB diagnostic tests such as a lateral flow assay. Several TB serum biomarkers have been identified and can be used for the diagnosis of TB. TB biomarkers expressed in serum samples were identified through in silico approach. The biomarkers were expressed in bacterial systems using recombinant DNA technology. The recombinant proteins were purified by affinity chromatography and further used as targets for the selection of aptamers using Systemic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers for the selected biomarkers were synthesized based on magnetic-bead based SELEX and characterized by electrophoretic mobility shift assay (EMSA), Surface Plasmon resonance (SPR) and MicroScale Thermophoresis (MST). Six putative TB serum biomarker proteins were selected from literature, namely, Insulin-like Growth Factor Binding Protein 6 (IGFBP6), Interferon-stimulated Gene 15 (ISG15), Calcium Binding Protein (S100A9), Retinol Binding Protein 4 (RBP4), Granzyme A (GrA), and Transgelin-2 (TAGLN2). The biomarkers were recombinantly expressed and purified after which they were used as targets in SELEX for aptamers synthesis. Aptamers were analysed by in silico method and the ones with highly conserved motifs were selected. The selected aptamers were synthesized and later characterized. The aptamers that show high affinity and specificity for the biomarkers will be used for the fabrication of a rapid lateral flow device for TB screening. Such a test would allow for a short diagnostic turnaround time, and hence expedite treatment.

Orientador: Valber de Albuquerque Pedrosa / Resumo: Neste trabalho foram desenvolvidas metodologias para detecção de biomarcadores cancerígenos utilizando sensores aptaméricos. Aptâmeros são estruturas tridimensionais de DNA/RNA capazes de serem seletivos a alvos específicos. O uso destas sequências em plataformas eletroquímicas permite que o monitoramento do metabolismo celular se torne viável de uma forma rápida e exata. Para a caracterização das superfícies foram utilizadas as técnicas eletroquímicas. Foram utilizadas três proteínas biomarcadoras, PSA, fPSA e HK2 como modelos para os estudos. Os limites de detecção do aptasensor para PSA e fPSA obtidos foram de 1,1 ng/mL e 2,9 ng/mL, respectivamente. Ao final dos experimentos com linhagens celulares cancerígenas e controle, as correspondentes corroboraram com a classificação de risco do câncer de próstata bem como a capacidade de diferenciação por parte dos aptasensores entre os tipos celulares mais agressivos (PC-3, [PSA]: 23 ng/mL; [fPSA]: 6 ng/mL) e menos agressivos (LNCaP, [PSA]:12 ng/mL; [fPSA]: 7,8 ng/mL) em comparação aos grupos de referência (PNT-2, [PSA]: 1,95ng/mL; [fPSA]: 2,11 ng/mL). Para a HK2, foi possível detecta-lá na presença de todos os tipos celulares prostáticos de maneira qualitativa. / Mestre