Molecular diagnostics is a fast growing field with new technologies being developed constantly. There is a demand for more sophisticated molecular tools able to detect a multitude of molecules on a single molecule level with high specificity, able to distinguish them from other similar molecules. This becomes very important for infectious diagnostics with the increasing antibiotic resistant viruses and bacteria, in gene based diagnostics and for early detection and more targeted treatments of cancer. For increased sensitivity, simplicity, speed and user friendliness, novel readouts are emerging, taking advantage of new technologies being discovered in the field of nanotechnology.  This thesis, based upon four papers, examines two novel electrical readouts for amplified single molecule detection. Target probing is based upon the highly specific amplification technique rolling circle amplification (RCA). RCA enables localized amplification resulting in a long single stranded DNA molecule containing tandem repeats of the probing sequence as product. Paper I demonstrates sensitive detection of bacterial genomic DNA using a magnetic nanoparticles-based substrate-free method where as few as 50 bacteria can be detected. Paper II illustrates a new sensor concept based on the formation of conducting molecular nanowires forming a low resistance circuit. The rolling circle products are stretched to bridge an electrode gap and upon metallization the resistance drops by several orders of magnitude, resulting in an extremely high signal to noise ratio. Paper III explores a novel metallization technique, demonstrating the efficient incorporation of boranephosphonate modified nucleotides during RCA.  In the presence of a silver ion solution, defined metal nanoparticles are formed along the DNA molecule with high spatial specificity. Paper IV demonstrates the ability to manipulate rolling circle products by dielectrophoresis. In the presence of a high AC electric field the rolling circle products stretch to bridge a 10 µm electrode gap.

Cette thèse présente le développement d'une nouvelle méthode de détection portable d'ADN bactérien. Une solution concentrée de longs ADNs forme des agrégats lorsqu'elle est soumise à un fort champ électrique dans un microcanal, qui sont détectables par conductimétrie. Après optimisation, un instrument portable incluant un système microfluidique et un module électronique compact pour le contrôle des champs électriques a été obtenu. Pour la détection de biomarqueurs usuels, une amplification isotherme d'ADN appelée HRCA a été testé de façon à allonger puis amplifier ces courts ADN en longs produits. Des expériences en temps-réel, une simulation de la cinétique d'amplification et une modélisation théorique ont permis la caractérisation de la réaction et de conclure que la plupart des produits générés en solution sont de petites tailles. Une réaction en deux étapes combinant une SDA et une RCA a finalement été développée pour amplifier, allonger puis détecter 10pM de séquences d'ADN issues de Staphylococcus Aureus. L'intégration sur puce d'une étape de traitement de l'échantillon et de la réaction d'amplification a nécessité le développement d'une nouvelle technologie nommée "murs mobiles". Ces structures 3D, contenant des microcanaux ou des membranes en hydrogel, peuvent être déplacées manuellement à l'intérieur de microsystèmes pour diverses applications: valves, séparation de chambres microfluidiques, préconcentration de molécules ou pour le développement de puces microfluidiques reconfigurables. / This thesis presents the development of a new portable detection method for bacteria. When subjected to a high electric field in a confined microchannel, a concentrated solution of long DNA formed large aggregates, detectable by conductometry. A previous prototype has been optimized to obtain a portable and label-free instrument including a microfluidic system and a compact electronic for the control of the electric field. To apply this technique to current DNA biomarkers, an isothermal amplification reaction called Hyper-branched Rolling-Circle Amplification (HRCA) was tested in order to simultaneously elongate and amplify short DNAs into long products. Real-time experiments, kinetic simulations and theoretical approaches were used to characterize and model this reaction, leading to the conclusion that most of the products generated in solution are small DNAs. A two-step amplification combining a Strand-Displacement Amplification (SDA) and a RCA was finally developed to amplify then elongate DNA sequences from Staphylococcus Aureus, with a LOD of 1pM. Preconcentration of DNA and addition of reagents for sequential reactions into a microreactor were necessary to integrate pre-detection steps into a portable system. A new toolbox, called "sliding wall" was developed for these needs. Elongated microfabricated structures containing channels or hydrogel membranes could be manually displaced into a closed microchip, enabling compartmentalization, valving, molecule trapping and reconfiguration of a channel network.

Nanopore sensors are emerging as a promising technology for single molecule analysis and polymer sequencing. Traditionally, measurements are taken by monitoring the ionic current through the nanopore, which gives information (e.g. size, shape, charge) about a molecule of interest while it is in the confined geometry of the nanopore. The dynamics of the molecule before the arrival to the nanopore, such as the capture dynamics, or molecular conformation prior to translocation, as well as clogging mechanisms and features of anomalous translocation events, are not assessed by the electrical measurements alone. To study the whole process of nanopore diffusion, capture and passage it is necessary to complement the electrical signal with another detection mode. Particularly, optical visualization of the molecules as they translocate through the nanopore has great potential. In this Thesis I present the design, construction, optimization and testing of a nanopore--‐based optofluidic instrument, which uses fluorescence microscopy to visualize individual fluorescently stained DNA molecules as they translocate a solid--‐state nanopore, while in parallel record the ionic current signal through the pore. The following challenges were overcome to achieve the integration of the optical and electrical systems: (i) the electrical detection system must account for the physical constrains of a wide field fluorescence microscope, and the optical system should in turn not affect the low--‐noise electrical detection of individual DNA molecules. The design of the instrument included a microfluidic device, so to position the nanopore within the working distance (<170--‐μm) of the microscope objective (Chapter 2). (ii) Electrical noise was optimized to a level that is indistinguishable from a standard (with no optics) nanopore system (Chapter 3). The custom instrument was used to demonstrate: 1) Electrical detection of DNA translocations with a laser light illuminating the nanopore; 2) Optical tracking of DNA capture and translocation dynamics; 3) Synchronization of the optical and electrical signals in preparation for simultaneous detection. In the process of noise optimization, a strong noise coupling between the illumination source and the ionic current was found, characterized and eliminated. Consequently, the noise performance of the custom instrument is the lowest of any other nanopore--‐based optofluidic systems described in the literature to date. This opens up the way to many new and exciting investigations of polymer translocation dynamics through nanoconfined geometries. Lastly, during the development of this custom instrument, a method to localize the fabrication of a nanopore by controlled dielectric breakdown on a membrane, with a focused laser beam, was discovered.

DNA detection has an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used not only in research laboratories, but also in clinical and forensic practice. The present thesis aims to unravel the potential of cellulose filter paper to be a viable candidate for DNA array support. There are two papers in this study. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on acitve paper using fluorescence. In Paper II, we investigated visualization and throughput of DNA detection with magnetic beads on active filter papers, an assay which requires no instrumentation (scanner). The findings in Paper I show that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups to detect DNA. The detection limit of the assay is approximately 0.2 pmol. In Paper II, visualization of DNA detection on active paper is achieved without instrumentation, based on the natural color of magnetic beads. Furthermore, successful multiplex detection supports the potential to increase the throughput of DNA detection on active papers. In summary, these studies show that active cellulose filter paper is a good DNA array support candidate as it provides a user-friendly and cost-efficient DNA detection assay. The methods described in Paper I and II are possible sources of development to a point-of-care device for on-site analysis of DNA contents in a sample. / <p>QC 20131111</p>

Magnetic nanoparticles have great potential in the biomedical and diagnostics field. Due to their small size, the particles have a high surface-to-volume ratio which enables for biofunctionalisation with different molecular probes. This makes itpossible to target them against a wide variety of biomarkers. In this project, the aim was to develop a magnetic nanoparticle-based DNA detection method with respectto sensitivity by employing circle-to-circle amplification, which is an extension of rolling circle amplification, in order to increase the assay sensitivity. The method provides high specificity due to the use of padlock probes for amplification. The project included testing and optimising the protocol used for DNA amplification and detection with a synthetic target, which involved testing different padlock probes, incubation times and incubation temperatures. Lastly, the method was tested on a biological target. It has recently been shown that specific aggregation occurs between magnetic nanoparticles and DNA, which enables for a visual readout strategy sincethe aggregates are visible to the naked eye. Initial testing of the method yielded asensitivity of about 100 attomoles. The achieved sensitivity after the optimisation work was 1 attomole of both synthetic and biological DNA targets. This is an improvement compared to the 400 attomoles that has previously been reported with one round of rolling circle amplification. The results can be used in further development of the naked-eye DNA detection method towards the realisation of a commercially attractive bioanalytical device.

Les biocapteurs ont pour objectif de détecter de faible quantité de biomolécules afin d'améliorer laqualité et la précocité des diagnostics médicaux. Parmi eux, les transistors à nanofils sont desdispositifs prometteurs, car ils permettent la détection électrique de biomolécules sans marquage avecune grande sensibilité et un temps de réponse court. Actuellement, la plupart de ces dispositifs utilisedes nanofils de silicium, qui peuvent être limités par une faible résistance chimique, ce qui entrainedes variations du signal en présence de solutions biologiques. Pour palier ces inconvénients, le carburede silicium (SiC) est un matériau prometteur déjà utilisé dans le domaine biomédical pour lafabrication ou le recouvrement de prothèses ou de vis médicales. Outre ses propriétés semiconductrices,ce matériau est biocompatible et montre une forte inertie chimique. Par conséquent, ilouvre une voie à l'intégration in-vivo des capteurs.L'objectif de cette thèse est d'élaborer des biocapteurs SiC à l'échelle nanométrique pour détecter desmolécules d'ADN. La première étape est la fabrication des transistors à base de nanofils SiC à grillearrière. Un procédé original de fonctionnalisation combiné avec la lithographie et aboutissant augreffage covalent de molécules sondes d'ADN a été mis au point. Finalement, la réponse des capteursa été mesurée entre chaque étape du protocole de fonctionnalisation. Les variations du signal lors desétapes de greffage et d'hybridation des molécules d'ADN démontrent la capacité de ces dispositifs àdétecter des molécules d'ADN. Des mesures complémentaires ont aussi montré la stabilité, lasélectivité et la réversibilité du dispositif. / Biosensors are designed to detect small quantities of biomolecules in order to improve the accuracyand earliness of medical diagnosis. Among them, nanowire transistors are promising devices, as theyallow the electrical detection of biomolecules without labeling with high sensitivity and a shortresponse time. Currently, most of these devices use silicon nanowires, which can be limited by a lowchemical resistance, which leads to signal variations in the presence of biological solutions. Toovercome these limitations, silicon carbide (SiC) is a promising material already used in thebiomedical field for the coating of prosthesis or bone screws. In addition to its semiconductingproperties, this material is biocompatible and shows a high chemical inertness. Therefore, it opens theway for in vivo integration of sensors.The goal of this thesis is to develop SiC biosensors at the nanoscale to detect DNA molecules. Thefirst step is the fabrication of SiC nanowire-based back gate transistors. A novel process combiningfunctionalization and lithography leading to the covalent grafting of DNA probe molecules has beendeveloped. Finally, the sensor response was measured between each step of the functionalizationprocess. The variations of the signal during the steps of grafting and hybridization of DNA moleculesdemonstrate the ability of these devices to detect DNA molecules. Additional steps have also shownthe stability, selectivity and reversibility of the device.

This bachelor’s thesis deals with design and optimalisation of custom biosensor for detection of methylated DNA. Teoretical part explains the mechanism and importance of DNA methylation. Next section describes analytical methods used in connection with DNA methylation and some basic direct and indirect methods of detection. Final part is dedicated to experiment itself, which is divided into several sections. Section one deals witch modification of working electrode and optimalisation of detection method. Second section introduces two different ways of DNA methylation detection. First is based on direct detection and second one on detection through the biosensor. Final part shows determination of methylcytosine from sample based on analysing characteristic attributes of signal and numeric algorithm based on curve fitting.

Despite the increasing number of electrochemical and photoelectrochemical biosensors reported in the research literature, few have achieved success outside of a laboratory setting. This can partly be attributed to accessibility issues with commercially available readout instruments. Consequently, low-cost and portable readout instruments have been developed by researchers, but these devices fail to address other key compatibility and accessibility challenges. Much like the commercial systems, these devices are not natively compatible with multiplexed signal assays consisting of two or more working electrodes, cannot control optical excitation sources for photoelectrochemical biosensing, nor can they interface with auxiliary instruments such as heaters and electromagnets. To this end, we have developed a low-cost smartphone-operated electrochemical and photoelectrochemical readout system for point-of-care biosensing. Our readout system can perform standard voltammetric techniques and is capable of synchronously controlling an optical excitation source to support photoelectrochemical biosensing. This device is compatible with standard three-electrode assays as well as dual signal assays with two working electrodes. We have also created a portable sample heater that can be controlled by this readout system to facilitate on-site sample heating and have also integrated a portable electromagnet to perform away-from-lab magnetic manipulation. / Thesis / Master of Applied Science (MASc) / Early and prompt detection of disease biomarkers is crucial in order to develop effective disease management strategies. Unfortunately, many gold-standard diagnostic techniques for infectious diseases, cancers, heart diseases, among other conditions prove to be time-consuming, costly, and reliant on trained professionals in a laboratory setting. Electrochemical and photoelectrochemical detection are two sensing modalities that show promising potential for point-of-care applications, as they are easily miniaturized, inexpensive, and can be used to detect both the presence of and the amount of analyte present. However, up until now, these sensing modalities have mostly been confined to research settings. To expedite the commercialization of such sensors and to facilitate their translation to point-of-care diagnostics, we have developed a low-cost smartphone-operated electrochemical and photoelectrochemical readout system. Through the integration of peripheral instruments including a sample heater, electromagnet, and optical excitation source, this system is compatible with a number of different biosensors.

Made available in DSpace on 2017-07-25T19:30:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-02-22 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / Sclerotinia sclerotiorum infect many plants, including crops of great economic importance as soybeans (Glycine max) and oilseed rape (Brassica sp.), responsible for great losses. The plant disease control is the most important practical objective of plant pathology, but the correct and rapid diagnosis are essential to define strategies for the diseases management. Molecular techniques are able to amplify specific fragments from small amounts of genetic material and powerful tools widely used in various areas, including the phytopathological diagnosis. Several techniques have been studied and designed for the amplification of nucleic acids, including the LAMP (Loop-mediated isothermal amplification), which has high specificity, sensitivity and is fast. The aim of this study was to develop and optimize a specific molecular test for S. sclerotiorum by LAMP, as well as its validation to oilseed pare and soybean seeds samples. A set of six primers was designed and evaluated for S. sclerotiorum sensitivity and specificity detection. The composition of the LAMP reaction was enhanced for real-time (SS-qLAMP) and direct analysis (SS-cLAMP). The DNA from 57 isolates of S. sclerotiorum, DNA from several other plant pathogens and DNA from different cultures was tested. The DNA of all isolates of S. sclerotiorum were detected but no the other DNA samples. When testing the limit of detection of reactions, a single copy detections was suggested. By SS-qLAMP two curves were generated which can be used to estimate the amount of mycelium and DNA of S. sclerotiorum present in the samples analyzed. Bothe developed tests (SS-qLAMP and SS-cLAMP) can be applied to several purposes, such as detection of the pathogen in plant, spore traps, soil and seeds samples. Using seed samples with different contamination level, the test was optimized for canola and soybean seeds, SS-qLAMP(Canola) and SS-qLAMP(Soybean), respectively, detecting the presence of the pathogen in samples up to 0.13% and 0.03% naturally contaminated for canola and soybean, respectively, and was able to detect contamination in samples not contaminated according incubation-based methods. The time require for the test was 4h and 30 minutes and 2 hours and 50 minutes for canola and soybeans, respectively, with no needs of large space for samples incubation, specialized analysts and able to analyzed many samples simultaneously. The proposed method SS-qLAMP was well-validated according the ISTA (International Seed Testing Association) rules to oilseed-rape and soybean seed samples. x / Sclerotinia sclerotiorum é um fungo que pode atacar diversas espécies vegetais, incluído culturas de grande importância econômica como soja (Glycine max) e canola (Brassica sp.), causando grandes prejuízos para as mesmas. O controle de doenças de plantas é o mais importante objetivo prático da Fitopatologia, mas, a correta e rápida diagnose da doença são pré-requisitos indispensáveis para definir as medidas para o manejo das mesmas. Técnicas moleculares capazes de amplificar fragmentos específicos a partir de pequenas quantidades de material genético são poderosas ferramentas amplamente utilizadas em diversas áreas, incluindo o diagnóstico fitopatológico. Diversas técnicas têm sido estudadas e criadas para a amplificação de ácidos nucleicos, entre elas a LAMP (Amplificação isotérmica mediada por “loops”), que apresenta alta especificidade, sensibilidade e é rápida. O objetivo do presente estudo foi desenvolver e otimizar um teste molecular específico para S. sclerotiorum a base de LAMP, com obtenção de resultados em tempo real e quantitativos (qLAMP) e análise visual direta de resultados (cLAMP), assim como a validação do mesmo para ser utilizado na análise de amostras de sementes de canola e soja. Um conjunto de seis primers foi desenhado e avaliado quanto a sensibilidade e especificidade de detecção de S. sclerotiorum. A composição da reação de LAMP foi otimizada quanto a concentração de diversos componentes tanto para a análise em tempo real como direta, compondo, respectivamente as reações de SS-qLAMP e SS-cLAMP, conforme necessário. Testou-se o DNA de 57 isolados de S. sclerotiorum, o DNA de diversos outros fitopatógenos e o DNA de diversas culturas. O DNA de todos os isolados de S. sclerotiorum foram detectados mas não o de outros fitopatógenos e de plantas. Ao testar o limite de detecção das reações, não houve um limite de detecção, sugerindo que a presença de qualquer molécula de DNA alvo seria detectada. Pelo método quantitativo foi possível gerar duas curvas pelas quais pode-se estimar a quantidade de micélio e de DNA de S. sclerotiorum presente na amostra analisada. Desenvolveu-se um teste específico para S. sclerotiorum a base de LAMP, denominada SS-LAMP que pode ser aplicado em diversos casos, como a detecção do patógeno em amostras de plantas, armadilhas de esporos, de solo e sementes. Otimizou-se o teste SS-LAMP para sementes de canola e soja, SS-qLAMP(Canola) e SS-qLAMP(Soja), respectivamente, detectando-se a presença do patógeno em amostras com até 0,13% e 0,03% de contaminação natural, para canola e soja, respectivamente, além da detecção em amostras que os métodos tradicionais não detectaram. O tempo total do método foi de 4h e 30 minutos para canola e 2h e 50 minutos para soja, sem a necessidade de amplo espaço para incubação das amostras, pessoal especializado para análise e com a possibilidade de diversas amostras serem analisadas concomitantemente. Assim validou-se o método proposto SS-qLAMP(Soja) segundo as regras da ISTA (International Seed Testing Association – Associação Internacional de Análise de Sementes).

Gold nanoshells are tunable plasmonic nanostructures consisting of spherical silica cores wrapped with thin layer of Au. Based on the size of the Au layer with respect to the silica core, gold nanoshells can resonantly absorb or scatter light at any wavelength on the visible or infrared. On resonance, gold nanoshells interact strongly with light to give rise to collective oscillations of the free electrons against the background of the ionic core, phenomena known as localized surface plasmons. The free electron oscillation creates surface plasmon multimodes of various orders. As a result, the average local near field surrounding the Au nanoshell is enhanced. The local field enhancement has been extensively used in different applications. In this work, the local near-field is used to enhance the Raman spectroscopy of DNA and explore the different modes attributed to the base composition and structure of the DNA sequence. We showed that urface enhanced Raman spectroscopy of DNA is dominated by the adenine modes regardless of the base composition of the DNA sequence, a property that we have used to develop a DNA label-free detection system. As absorbers, plasmon-resonant Au nanoshells can convert absorbed light into heat. As a consequence, the temperature on the Au nanoshell surface increases dramatically. This property is used to light-trigger the release of variety of therapeutic molecules such as single stranded DNA, siRNA and small molecules. We demonstrated that the local heat can be used to dehybridize double stranded DNA attached to the Au surface via a thiol moiety on one of the DNA strands. The complementary sequence (therapeutic sequence) is released at temperature lower than the standard melting temperature of same DNA sequence. Moreover, small molecules (DAPI) which were initially intercalated on the double stranded DNA attached to the Au surface were successfully released due to the heat generated around the nanoshell surface. Finally, siRNA molecules were also released using a different system made of PLL (polylysine) attached to Au nanoshells. The electrostatic interaction between the negatively charged siRNA and the positively charged PLL was overcome by the thermal perturbation causing the siRNA to be released. In vitro experiments successfully showed the release of siRNA, single stranded DNA and small molecules.