Development of single-particle counting assays with interferometric reflectance imaging
Ekiz Kanik, Fulya
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Biomarkers are biological measures used for clinical assessment, whether an individual has a particular medical condition or to monitor and predict health states in individuals. Sensitive detection and quantification of various biomarkers are essential for disease diagnostics. The majority of biomarker-based diagnostics examines the presence and quantity of a single biomarker. Since the symptoms of many diseases are alike, multiplexed biomarker tests are highly desirable. Furthermore, detection of multiple biomarkers would improve the accuracy of diagnosis as well as providing additional information about the prognosis. Microarray platforms have the potential for higher level of multiplexing for biomarker detection. However, conventional microarray technologies are limited by the sensitivity of assays. This dissertation describes how single-particle interferometric reflectance imaging sensor (SP-IRIS) overcomes the sensitivity issues in biomarker detection and its applications to biomolecular and cellular biomarker detection assays. SP-IRIS provides optical detection of individual nanoparticles when they are captured onto a simple reflecting substrate, providing single-molecule sensitivity. This technique can be used to detect natural nanoparticles (such as viruses) without labels as well as molecular analytes (proteins and nucleic acids) that are labeled with metallic nanoparticles. Moreover, the advancements in technology make SP-IRIS ideal for the detection of low abundance biomarkers. Utilization of light polarization in combination with plasmonic gold nanorods as labels enhances the signal-to-noise ratio in nanoparticle detection allowing for the use of low numerical aperture optics increasing the field-of-view, hence, the throughput and sensitivity. Additionally, the integration of a disposable microfluidic flow cell and dynamic particle tracking in kinetic measurements provide a robust, ultra-sensitive and automated diagnostic platform. This dissertation focuses on the development of biological assays demonstrating effective use of SP-IRIS as a clinical diagnostic platform. We discuss the development of protein, nucleic acid and biological nanoparticle detecting SP-IRIS microarrays. We demonstrate four digital detection platforms for Hepatitis B, microRNA, rare mutations in an oncogene, KRAS, and virus-like particle detection with ultra-high sensitivity.
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