Graduate Thesis Or Dissertation
 

Detection techniques for biomolecules using semi-conductor nanocrystals and magnetic beads as labels

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  • Continued interest in the development of miniaturized and portable analytical platforms necessitates the exploration of sensitive methods for the detection of trace analytes. Nanomaterials, on account of their unique physical and chemical properties, are not only able to overcome many limitations of traditional detection reagents but also enable the exploration of many new signal transduction technologies. This dissertation presents a series of investigations of alternative detection techniques for biomolecules, involving the use of semi-conductor nanocrystals and magnetic beads as labels. Initial research focused on the development of quantum dot-encapsulating liposomes as a novel fluorescent label for immunoassays. This hybrid nanomaterial was anticipated to overcome the drawbacks presented by traditional fluorophores as well as provide significant signal amplification. Quantum dot-encapsulating liposomes were synthesized by the method of thin film hydration and characterized. The utility of these composite nanostructures for bioanalysis was demonstrated. However, the long-term instability of the liposomes hampered quantitative development. A second approach for assay development exploited the ability of gold nanoparticles to quench the optical signals obtained from quantum dots. The goal of this study was to demonstrate the feasibility of using aptamer-linked nanostructures in FRET-based quenching for the detection of proteins. Thrombin was used as the model analyte in this study. Experimental parameters for the assay were optimized. The assay simply required the mixing of the sample with the reagents and could be completed in less than an hour. The limit of detection for thrombin by this method was 5 nM. This homogeneous assay can be easily adapted for the detection of a wide variety of biochemicals. The novel technique of ferromagnetic resonance generated in magnetic bead labels was explored for signal transduction. This inductive detection technique lends itself to miniaturization, is capable of mass production and is inexpensive to fabricate. The device consisted of a microwave circuit in which a slotline and a coplanar waveguide were integrated with a biochemically activated sensor area. The magnetic beads were immobilized at the sensor area by bio-specific reactions. Experiments conducted on this prototype show promising results for using ferromagnetic resonance-based detection of magnetic labels for fabrication of portable and inexpensive sensor devices. The next stage of work addresses the issue of patterning of sensing surfaces with biomolecules. The ability to selectively immobilize biomolecules on surfaces has far-reaching applications, including sensor development. A simple and widely applicable method for the photopatterning of chitosan films with biotin was presented. Chitosan is a biocompatible and biodegradable polymer. The proposed method was capable of forming spatially defined biotin features on the order of tens of microns, together with a significant reduction of non-specific protein binding and increase in hydrophilicity of the sensor surface. The entire patterning process, inclusive of the blocking step, could be completed in under an hour. This straightforward method for the selective patterning of the biocompatible polymer chitosan is expected to be widely useful in the field of bioanalysis.
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