The development of FRET-based dual receptor optical biosensor

The focus of the research presented in this dissertation is the development of a new FRET-based dual receptor sensing method for detecting the human immunodeficiency virus (HIV). The new detection method presented in this dissertation imitates the way HIV infects cells. It utilizes the two receptor-binding event and integrates a chemical transducer system with two unique protein receptors, CD4 and mAb (HIV-1 gp120 monoclonal antibody), which both bind to gp120. The chemical transduction system is based on the distance-dependant principle of fluorescence resonance energy transfer (FRET).; The work presented in this dissertation attempts to demonstrate the feasibility of this new sensing method both in solution and on an optical fiber. Appropriate FRET pairs which have high energy transfer efficiency as well as good conjugation properties with receptors were selected and optimized. The two receptors, CD4 and mAb which specifically bind to gp120, were conjugated to one of the optimized FRET fluorophore pairs, AMCA-NHS (succinimidyl-7-amino-4-methylcoumarin-3-acetic acid) and FITC (fluorescein isothiocyanate), respectively. For the solution test, the viral protein gp120, which is the featured protein on the surface of HIV-1, was detected by the mixed solution of the two FRET pair tagged receptors. A spectrofluorometer was used to detect the fluorescent change between AMCA-NHS and FITC peak intensities when the receptors bind to the gp120. Specific binding and non-specific binding gp120 were used to test the selectivity of this method. The results of the solution test indicated that FRET-conjugated receptors can efficiently distinguish the presence of specific and non-specific binding gp120 and proved the feasibility of the FRET-based dual receptor method in detecting the presence of gp120 with a limit of detection of 5ng/ml (0.5nM) in solution. For the optical fiber test, two FRET-conjugated receptors were immobilized onto an optical fiber silica core tip to detect the presence of gp120 around its surface. The test results between two individual receptors and gp120 demonstrated that the two FRET pair tagged receptors could be efficiently immobilized onto a silanized fiber tip without losing their affinities for gp120. (Abstract shortened by UMI.)...