| This thesis details research toward the development of a single chain variable fragment fiber-optic (scFv) microarray. The intended purpose of this scFv-based microarray is use as a salivary based diagnostics tool. Single chain variable fragment based microarrays expand upon established antibody based microarrays and offer unique advantages such as high sensitivity and availability of high affinity scFv reagents. Chapter 1 describes the use of saliva as a diagnostic medium and the use of protein biomarkers for the diagnosis of disease states. Chapter 2 covers fundamental chemistry, theory, and mechanics of the fiber-optic microarray platform. Chapter 3 introduces the concepts of immunoassays as well as discusses the architecture of both IgG antibodies and single chain variable fragments. Understanding of both the structure of scFvs and immunoassays will aid in grasping the methodology and implementation of scFv reagents into bead-based fiber-optic microarrays. Chapter 4 presents all relevant data supporting the development and function of scFv-based fiber-optic microarrays. All scFv reagents were graciously provided by Dr. Mark Sullivan from the University of Rochester. ScFv-based microarrays developed in the Walt laboratory for the detection of salivary alpha-amylase exhibit a limit of detection of 1 nM alpha-amylase. A three point calibration curve was constructed and linear regression was performed. From these data, the equation (y+/-3.6) = (0.17+/-0.02)x + (27+/-2. 8) is obtained, where y is the measured fluorescence intensity and x is the concentration of alpha-amylase. |
