Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Engineering Science

Program

Biomedical Engineering

Supervisor

Zhang, Jin

Abstract

Carbon dot (CD) is a recently discovered fluorescent nanoparticle that exhibits excellent optical properties while also displaying great biocompatibility. This thesis examines two applications of CD: development of self-illuminating CD and investigation of the ability of CD in crossing biological barriers.

The self-illumination of CD is achieved through bioluminescence resonance energy transfer (BRET) from a bioluminescent protein, Renilla luciferase, to CD. The synthesis of self-illuminating CD consisted of using N’-(3-dimethylamnopropyl)-N-ethylcarbodiimide as coupler with 0.20 mg/mL to 0.80 mg/mL of CD and incubating with 2 uM Rluc for 6.5 hours. The BRET efficiency increased with increasing CD concentration.

Bovine blood retina barrier (BRB), was used a biological barrier model. CD was incubated inside the eye after the removal of the vitreous. Preliminary data suggests that CD can cross the BRB as CD was found in the retinal neural layer of BRB as well as the choroid layer of BRB after treatment.

Summary for Lay Audience

Carbon dot (CD) is a nano sized synthetic material that was recently discovered. Because CD can fluoresce brightly, it has been mostly used to substitute less bright traditional dyes in bioimaging. As CD was recently discovered, the potential applications of CD have not been investigated thoroughly. The thesis consists of two studies: development of self-illuminating carbon dot and investigation if CD can cross biological barriers.

Although CD can fluoresce brightly, the major downside is that an external light source has to excite CD for its fluorescence. The external light source can result in inaccuracy in results and damage to samples as the light is very powerful. Self-illumination was achieved by conjugating a bioluminescent protein, Renilla luciferase (Rluc), to CD. Rluc fluoresces when its substrate, coelenterazine, is present; the fluorescence from Rluc can excite CD so that it fluoresces. Such mechanism is referred to as bioluminescence resonance energy transfer (BRET). The study found that the synthesis of self-illuminating CD consists of a coupler, N’-(3-dimethylamnopropyl)-N-ethylcarbodiimide, to help with the conjugation, 0.2 mg/mL to 0.8 mg/mL of CD and incubating it with 2uM of Rluc for 6.5 hours. The BRET efficiency was found to increase with increasing CD concentrations. Self-illumination eliminates the disadvantages of external light source while maintaining the bright fluorescence of CD.

Biological barriers are physical barriers that exists to closely monitor what goes in and out of the most important parts of the body such as the brain and the eye; only extremely small or very specific materials can therefore enter and exit the biological barriers. Because it is easier to access intact eye structure compared to the brain, cow eyes were used to represent biological barriers. After incubating CD inside the eyes, both the retinal neural layer, the inside layer of the eye, and the choroid layer, the outside layer of the eye, had fluorescence after treatment, which suggests that CD might be able to cross BRB. With CD potentially crossing BRB, further studies can be done within the medical field to investigate and treat diseases that occur across the barriers.

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