Enabling and understanding nanoparticle surface binding assays with interferometric imaging
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There is great need of robust and high throughput techniques for accurately measuring the concentration of nanoparticles in a solution. Microarray imaging techniques using widely used to quantify the binding of labeled analytes to a functionalized surface. However, most approaches require the combined output of many individual binding events to produce a measurable signal, which limits the sensitivity of such assays at low sample concentrations. Although a number of high-NA optical techniques have demonstrated the capability of imaging individual nanoparticles, these approaches have not been adopted for diagnostics due complex instrumentation and low assay throughput. Alternatively, interferometric imaging techniques based on light scattering have demonstrated the potential for single nanoparticle detection on a robust and inexpensive platform. This dissertation focuses on the development of methods and infrastructure to enable the development of diagnostic assays using the Single Particle Interferometric Imaging Sensor (SP-IRIS). SP-IRIS uses a bright-field reflectance microscope to image microarrays immobilized on a simple reflective substrate, which acts as a common-path homodyne interferometer to enhance the visibility of nanoparticles captured near its surface. This technique can be used to detect natural nanoparticles (such as viruses and exosomes) as well as molecular analytes (proteins and nucleic acid sequences) which have been tagged with metallic nanoparticle in a sandwich assay format. Although previous research efforts have demonstrated the potential for SP-IRIS assays in a variety of applications, these studies have largely been focused on demonstrating theoretical proof of concept in a laboratory setting. In contrast, the effective use of SP-IRIS as a clinical diagnostic platform will require significant functional improvements in automation of assay incubation, instrument control, and image analysis. In this dissertation, we discuss the development of instrumentation and software to support the translation of SP-IRIS from manual laboratory technique into an automated diagnostic platform. We first present a collection of mechanical solutions to enable the real-time, in-solution imaging of nanoparticles in disposable microfluidic cartridges. Next, we present image analysis techniques for the detection of nanoparticle signatures within digital images, and discuss solutions to the unique obstacles presented by the ill-defined focal properties of homodyne interferometry. Finally, we present a particle tracking algorithm for residence time analysis of nanoparticle binding in real-time datasets. Collectively, these improvements represent significant progress towards the use of SP-IRIS as a robust and automated diagnostic platform.
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