An in-fibre biotin-streptavidin study
Tan, Wai Keong
Date of Issue2014
School of Chemical and Biomedical Engineering
Biosensors have played a crucial role in medical diagnostic, medical research, genetic and proteomic studies. With the change in global demographics and possible disease outbreak in areas with poor healthcare, the need for portable diagnostic device has become of paramount importance. With the development of microfluidics and microelectronic, it has made miniaturizing of these medical diagnostic tools possible and the advantages of using these miniaturised device are low cost in product, shorter waiting time and small of volume of test sample is required. For the past few decades, there have been an increased research interests in using optic fibre and photonic crystal fibre as possible biosensor due to its small size and light guiding ability using total internal refraction and photonic band gap has the potential to be used for biomolecule detection. In this proposed project, an in-fibre coating of streptavidin was carried out and later on study with the use of Biotin-4-Fluorescein (B4F). Streptavidin and biotin interaction is one of the strongest interaction with a dissociation constant (Kd) of 10-15 M identified and well documented. Linker molecules poly-L-lysine and gluaraldehyde have been used to immobilise streptavidin molecules in the fibre through charge interactions as shown in figure 9. Ethanolamine was added as a filler to block unbound glutaraldehyde site to prevent unwanted binding. During the studying of coated streptavidin interaction, B4F was introduced into the coated fibre and the fluorescence emission intensity was recorded over a period of 20 minutes. From the result obtained, a decreased emission intensity and increased excitation intensity was observed. These results obtained were desired as B4F would be quenched upon binding to streptavidin, reducing emission intensity and absorbance which is reflected by an increased in excitation intensity. Also from the PBS reading before and after B4F introduction, a shoulder was formed on the right of the curve and this indicates that there is streptavidin-B4F interaction. This result was made in comparison with uncoated fibre PBS reading before and after B4F introduction where there was no protrusion seen on the after B4F curve. With that, it can be concluded that streptavidin was successfully coated onto the interior of the fibre. The future work would be to fine tune the coating procedures and testing with other common protein and analytes.
Final Year Project (FYP)
Nanyang Technological University