Master of Engineering Science
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.
Summary for Lay Audience
Type 1 diabetes is a disease characterized by loss of blood glucose control due to autoimmune destruction of pancreatic beta islet cells. Modern treatment requires the use of a blood glucose meter, often requiring the pricking of the finger. Constant pricking is a detriment to the quality of life to these patients and may lead to decreases in patient adherence. The goal of this project is to develop a non-invasive alternative to measuring blood glucose. A newly designed sensor using a technology known as bioluminescence resonance energy transfer (BRET) has been developed with the hopes that it may replace traditional glucose sensing methods. BRET occurs when a light emitting protein transfers light energy to another light emitting protein. The second protein will then emit its own light, which can then be measured. The newly designed sensor uses quantum dots in the place of a second protein due to unique properties that make it suitable for glucose sensing. The sensor components are separated by a glucose binding protein, which changes conformation upon binding glucose. Therefore, the amount of light emitted by the quantum dot will correspond to whether glucose has bound the sensor. Testing of the sensor revealed that a 0.3125:1 ratio of the protein to quantum dots to be the most optimal. In addition, the sensor could detect variations of glucose levels ranging between 2µM to 0.1M, which is sufficient to detect levels of glucose in both tear samples and blood samples. The sensor was also tested while deposited on a solid substrate. The results indicate the sensor may be promising as an alternative to traditional blood glucose sensing.
Hwang, Eugene, "Bioluminescence resonance energy transfer (BRET) - based nanostructured biosensor for detection of glucose" (2019). Electronic Thesis and Dissertation Repository. 6782.
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