Date of Completion

Spring 5-1-2021

Thesis Advisor(s)

Ping Yan; James Cole

Honors Major

Structural Biology and Biophysics

Second Honors Major

Molecular and Cell Biology

Abstract

Voltage sensitive dyes (VSDs) are voltage reporters which are used to measure electrical activity in cells [1]. Tethered VSDs are a new class of VSD which consist of a flu- orophore and quencher component attached via hydrocarbon chain that is approximately the same length of a cell membrane. These dyes demonstrate potential to overcome the issues of poor fluorescence change in response to a changing electrical potential (low sensitivity) and insufficient speed that current VSDs exhibit while measuring electrical activity of a cellular membrane[1]. While tethered dyes have been able to demonstrate high levels of sensitivity in an in-vitro system [1], further optimization of fluorophore-quencher combinations will allow for the measurement of electrical activity in the near-IR (NIR) region with enhanced sensitivity, resulting in high quality recordings with significantly less equipment and labor required. Three different fluorescent molecules have been identified as possible tethered VSD components, these being a modified Fluorescein based molecule, a DiSBAC(5) [Bis-(1,3-dibutylthiobarbituric acid) pentamethine oxonol] molecule, and a modified Cy-5 molecule. In-vitro measurements of an induced changing electrical field have resulted in sensitivity measurements of up to 11%/100 mV fluorescence change for a DiSBAC(5) molecule in a two-component system with a previ- ously synthesized di-4-ANEPPS molecule. Modifications to structure, fluorophore/quencher combinations, as well as tethering the previously mentioned molecules are likely to strengthen these sensitivity measurements further making their use for an in-vivo system more feasible.

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