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APPLICATIONS OF MICROBEAD-BASED ELECTROCHEMICAL IMMUNOASSAYTHOMAS, JENNIFER HODGES January 2003 (has links)
No description available.
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New micropatterning techniques for the spatial addressable immobilization of proteinsFilipponi, Luisa, n/a January 2006 (has links)
Bio-microdevices are miniaturised devices based on biologically derived components
(e.g., DNA, proteins, and cells) combined or integrated with microfabricated substrates.
These devices are of interest for numerous applications, ranging from drug discovery, to
environmental monitoring, to tissue engineering. Before a bio-microdevice can be fully
developed, specific fabrication issues need to be addressed. One of the most important
is the spatial immobilization of selected biomolecules in specific micro-areas of the
device. Among the biomolecules of interest, the controlled immobilization of proteins to
surfaces is particularly challenging due to the complexity of these macromolecules and
their tendency to lose bioactivity during the immobilization step. The present Thesis
reports on three novel micropatterning techniques for the spatial immobilization of
proteins with bioactivity retention and improved read-out of the resulting micropatterns.
The technologies developed are based on three different micropatterning approaches,
namely 1) direct-writing UV laser microablation (proLAB), 2) a novel microcontact
printing method (�CPTA) and 3) a replica molding method combined with bead selfassembly
(BeadMicroArray). The first two technologies, proLAB and �CPTA, are an
implementation of existing techniques (laser ablation and �CP, respectively), whereas
the third, i.e., the BeadMicroArray, is a totally new technique and type of patterning
platform.
'ProLAB' is a technology that uses a micro-dissection tool equipped with a UV laser
(the LaserScissors�) for ablating a substrate made of a layer of ablatable material, gold,
deposited over a thin polymer layer. The latter layer is transparent to the laser but
favours protein adsorption. In the present work microchannels were chosen as the
structure of interest with the aim of arranging them in 'bar-codes', so to create an
'information-addressable' microarray. This platform was fabricated and its application
to specific antigen binding demonstrated.
The second technique that was developed is a microstamping method which exploits the
instability of a high-aspect ratio rubber stamp fabricated via soft-lithography. The
technique is denominated microcontact printing trapping air (�CPTA) since the collapsing of a rubber stamp made of an array of micro-pillars over a plane glass surface
resulted in the formation of a large air gap around the entire array. The method can be
successfully employed for printing micro-arrays of proteins, maintaining biological
activity. The technique was compared with robotic spotting and found that microarrays
obtained with the �CPTA method were more homogeneous and had a higher signal-tonoise
ratio.
The third technique developed, the BeadMicroArray, introduces a totally new platform
for the spatial addressable immobilization of proteins. It combines replica molding with
microbead self-assembling, resulting in a platform where diagnostic beads are entrapped
at the tip of micropillars arranged in a microarray format. The fabrication of the
BeadMicroArray involves depositing functional microbeads in an array of V-shaped
wells using spin coating. The deposition is totally random, and conditions were
optimised to fill about half the array during spin coating. After replica molding, the
resulting polymer mold contains pyramid-shaped posts with beads entrapped at the very
tip of the post. Thanks to the fabrication mode involved, every BeadMicroArray
fabricated contains a unique geometric code, therefore assigning a specific code to each
microarray. In the present work it was demonstrated that the functionality of the beads
after replica molding remains intact, and that proteins can be selectively immobilized on
the beads, for instance via biorecognition. The platform showed a remarkable level of
selectively which, together with an efficient blocking towards protein non-specific
adsorption, lead to a read-out characterized by a very good signal-to-noise. Also, after
recognition, a code was clearly visible, therefore showing the encoding capacity of this
unique microarray.
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Optimization and Ultimate Limitations for Immunoassay and Clinical DiagnosticsJanuary 2015 (has links)
abstract: Biological fluids, in particular blood plasma, provide a vital source of information on the state of human health. While specific detection of biomarker species can aid in disease diagnostics, the complexity of plasma makes analysis challenging. Despite the challenge of complex sample analysis, biomarker quantification has become a primary interest in biomedical analysis. Due to the extremely specific interaction between antibody and analyte, immunoassays are attractive for the analysis of these samples and have gained popularity since their initial introduction several decades ago. Current limitations to diagnostics through blood testing include long incubation times, interference from non-specific binding, and the requirement for specialized instrumentation and personnel. Optimizing the features of immunoassay for diagnostic testing and biomarker quantification would enable early and accurate detection of disease and afford rapid intervention, potentially improving patient outcomes. Improving the limit of quantitation for immunoassay has been the primary goal of many diverse experimental platforms. While the ability to accurately quantify low abundance species in a complex biological sample is of the utmost importance in diagnostic testing, models illustrating experimental limitations have relied on mathematical fittings, which cannot be directly related to finite analytical limits or fundamental relationships. By creating models based on the law of mass action, it is demonstrated that fundamental limitations are imposed by molecular shot noise, creating a finite statistical limitation to quantitative abilities. Regardless of sample volume, 131 molecules are necessary for quantitation to take place with acceptable levels of uncertainty. Understanding the fundamental limitations of the technique can aid in the design of immunoassay platforms, and assess progress toward the development of optimal diagnostic testing. A sandwich-type immunoassay was developed and tested on three separate human protein targets: myoglobin, heart-type fatty acid binding protein, and cardiac troponin I, achieving superior limits of quantitation approaching ultimate limitations. Furthermore, this approach is compatible with upstream sample separation methods, enabling the isolation of target molecules from a complex biological sample. Isolation of target species prior to analysis allows for the multiplex detection of biomarker panels in a microscale device, making the full optimization of immunoassay techniques possible for clinical diagnostics. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015
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Effect of dissolved chlorine on an MS2 bacteriophage immunoassay and tryptophan side chainConklin, Natasha Mwale 21 July 2009 (has links)
No description available.
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Physics and Applications of Interacting Magnetic Particles: Effect of Patterned TrapsPrikockis, Michael Vito 08 June 2016 (has links)
No description available.
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Microfluidics for bioanalytical research : transitioning into point-of-care diagnosticsScida, Karen 09 February 2015 (has links)
In this dissertation, three different microfluidic devices with bioanalytical applications are presented. From chapter to chapter, the bioanalytical focus will gradually become the development of a point-of-care sensor platform able to yield a reliable and quantitative response in the presence of the desired target. The first device consists of photolithographically-patterned gold on glass bipolar electrodes and PDMS Y-shaped microchannels for the controlled enrichment, separation from a mixture, and delivery of two charged dyes into separate receiving microchannels. The principle for the permanent separation of these dyes is based on the concept of bipolar electrochemistry and depended on the balancing/unbalancing of convective and electromigrating forces caused by the application of a potential bias, as well as the activation/deactivation of the bipolar electrodes. Two different bipolar electrode configurations are described and fluorescence is used to optimize their efficiency, speed, and cleanliness of delivery. The second device is a DNA sensor fabricated on paper by wax printing and folding to form 3D channels. DNA is detected by strand-displacement induced fluorescence of a single-stranded DNA. A multiplexed version of this sensor is also shown where the experiment results in “OR” and “AND” Boolean logic gate operations. In addition, the nonspecific adsorption of the reagents to cellulose is studied, demonstrating that significant reduction of nonspecific adsorption and increased sensitivity can be achieved by pre-treating the substrate with bovine serum albumin and by preparing all analyte solutions with spectator DNA. The third device, also made of paper, has a novel design and uses a versatile electrochemical detection method for the indirect detection of analytes via the direct detection of AgNP labels. A proof-of-concept experiment is shown where streptavidin-coated magnetic microbeads and biotin-coated AgNPs are used to form a composite model analyte. The paper device, called oSlip, and electrochemical method used are easily coupled so the resulting sensor has a simple user-device interface. LODs of 767 fM are achieved while retaining high reproducibility and efficiency. The fourth device is the updated version of the oSlip. In this case, the objective is to show the current progress and limitations in the detection of real analytes using the oSlip device. A sandwich-type immunoassay approach is used to detect human chorionic gonadotrophin (pregnancy hormone) present in human urine. Various optimization steps are performed to obtain the ideal reagent concentrations and incubation time necessary to form the immunocomposite in one step, that is, by mixing all reagents at the same time in the oSlip. Additionally, improvements to the electrochemical detection step are demonstrated. / text
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Cytokine capture with beads in cytotoxicity assays in microwells / Cytokinfångning med kulor i cytotoxicitetsanalyser i mikrobrunnarSimon, Maxime January 2023 (has links)
Cytokines are small, secreted proteins that are important for cell signalling in theimmune system. Interferon gamma (IFN-γ) is one of the most potent cytokines thatnatural killer (NK) cells of the innate immune system secrete with both antiviral,antibacterial, and antitumoral activity. Analysis of NK cells, such as that of secretionof IFN-γ, is important for studying the immune response to cancer and for developingeffective immunotherapies. In this master thesis project, a method was developedfor determining the amount of IFN-γ secreted by NK cells when being confinedwith cancer cells in deep microwells. Antibody-coated microbeads was used tocapture secreted IFN-γ, which was fluorescently labeled and detected by imaging usingfluorescence microscopy. Microbead seeding into small microwells for single cellassays and into large microwells for embedding of beads into 3D tumor spheroidswas investigated. An analytical model based on experimental standard curves wasdeveloped for straightforward quantification of the amount of bound IFN-γ, with ademonstrated detection down to 2.10−18 moles per bead. The detection of IFN-γ wasevaluated for primary NK cells stimulated by PMA/ionomycin for different incubationtimes. The secretion rate of IFN-γ by IL-2 activated NK cells under PMA/ionomycinstimulation was estimated at 184 molecules per second. IFN-γ detection was alsoevaluated in cell cytotoxicity assays where NK cells were confined over time togetherwith cancer cells in microwells. Both assays showed a successful detection of IFN-γ secretion, demonstrating the potential of the developed method for immune cellanalysis. / Cytokiner är små proteiner som är viktiga för cellsignalering inom immunförsvaret.Interferon gamma (IFN-γ) är en av de mest potenta cytokinerna som naturligamördarceller (NK) i det medfödda immunsystemet utsöndrar med både antiviral,antibakteriell och antitumoral aktivitet. Analys av NK-celler, av till exempelutsöndring av IFN-γ, är viktigt för att studera immunsvaret vid cancer och för attutveckla effektiva immunterapier. I detta examensarbete har en metod utvecklatsför att bestämma mängden IFN-γ som utsöndras av NK-celler när de är tillsammansmed cancerceller i djupa mikrobrunnar. Antikroppsbelagda mikrokulor användesför att fånga utsöndrat IFN-γ, som sedan fluorescensinmärktes och detekteradesgenom fluorescensmikroskopi. Distributionen av dessa kulor studerades i småmikrobrunnar för encellsanalyser och i stora mikrobrunnar för inbäddning av kulornai 3D-tumörsfäroider. En analytisk modell baserad på experimentella standardkurvorutvecklades för enkel kvantifiering av mängden bunden IFN-γ, med en påvisaddetektion ner till 2.10−18 mol per kula. Detektionen av IFN-γ utvärderades för primäraNK-celler stimulerade med PMA/ionomycin för olika inkubationstider. Sekretionenav IFN-γ från IL-2-aktiverade NK-celler vid stimulering med PMA/ionomycinuppskattades till 184 molekyler per sekund. IFN-γ-detektion utvärderades ocksåför analyser av cell-cytotoxicitet där NK-celler var placerade tillsammans medcancerceller i mikrobrunnar över tid. Båda analyserna visade en framgångsrikdetektering av utsöndrad IFN-γ, vilket visar potentialen hos den utvecklade metodenför immuncellsanalys.
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Développement de réseaux multiplexés de biocapteurs électrochimiquesDeiss, Frédérique 20 November 2009 (has links)
Ce travail de thèse a porté sur le développement de réseaux de micro- et nanocapteurs opto-électrochimiques pour la bioanalyse. Ils répondent à la demande grandissante dans le domaine de la recherche et du diagnostic pour des outils permettant de réaliser de multiples analyses simultanément avec des échantillons de faibles volumes. Ces nouvelles biopuces de haute densité sont fabriquées à partir de faisceaux cohérents de fibres optiques. Une des deux faces est micro- ou nanostructurée par une attaque chimique, puis fonctionnalisée avec une sonde biologique. La première biopuce est un réseau de nanocapteurs fluorescents à ADN où les sondes ont été immobilisées grâce aux propriétés d’électropolymérisation du pyrrole. La lecture est réalisée à distance au travers du faisceau d’imagerie. En combinant la technique d’immobilisation avec des microleviers électrochimiques, plusieurs sondes différentes ont pu être adressées sur le même réseau nanostructuré. La seconde biopuce permet d’effectuer des immunodosages multiplexés en utilisant l’imagerie électrochimiluminescente résolue à l’échelle d’une microsphère. Le développement de cette technique permet de combiner les avantages de l’électrochimiluminescence avec des immunodosages multiplexés. L’élaboration de ces réseaux allie différentes techniques physico-chimiques, notamment électrochimiques, pour obtenir des biopuces avec un fort potentiel, grâce à une densité et un degré de multiplexage importants. / This work presents the development of optoelectrochemical micro- and nanosensor arrays for bioanalytical applications. These platforms respond to the growing need in research and diagnostic for tools allowing multiple and simultaneous analysis in small-volume samples. These new high density biochips are made from coherent optical fiber bundles: one face is micro- or nanostructured by chemical etching and then functionnalized with biological probes. The first biochip is a fluorescent DNA nanosensor array where probes have been immobilized by electrodeposition of a polypyrrole thin film. The detection of the hybridization is remotely performed through the imaging fiber. Different probes were succesfully addressed onto the same nanostructured array thanks to electrochemical cantilevers. The second biochip allows multiplexed sandwich immunoassays using electrochimiluminescent imaging resolved at the single bead level. In particular, the development of this new readout mechanism allows extending electrochemiluminescent detection for multiplexed immunoassays. Design and implementations of both platforms take advantages of different physical and chemical techniques, especially electrochemical, to obtain biochips with a great potential through high density and high multiplexing level.
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