Bioluminescence resonance energy transfer (BRET) - based nanostructured biosensor for detection of glucose
Abstract
Bioluminescence resonance energy transfer (BRET) is a distance dependent, non-radiative energy transfer, which uses a bioluminescent protein to excite an acceptor through resonance energy transfer. In this thesis, BRET technology is incorporated into a sensor comprised of a recombinant protein and quantum dots. The recombinant protein, which includes the bioluminescent protein, Renilla luciferase (Rluc), is used as the donor molecule and cadmium tellurium quantum dots as the acceptor molecules. Separating the donor-acceptor pair is a recombinant protein, glucose binding protein, which changes conformation upon binding glucose and brings the pair closer together, thus allowing BRET to occur. Optimization of the BRET sensor was investigated by evaluating different ratios of the donor and acceptor, changes in the bioconjugation process, and different glucose concentrations. The intensity of bioluminescence is a function of the ratio between the quantum dots to protein, which ranges from 1:6 to 0.0156:1, EDC ratio to quantum dots, conjugation time, and concentration of glucose ranging from 2μM to 0.1M. In addition, the performance of the sensor on a solid substrate was evaluated. This sensor promises to offer an alternative to traditional blood glucose sensing.