The overall goal of this project is to develop a biosensor for a variety of clinically relevant small molecules. Our current work focuses on the formation of a biosensor for the detection of tryptophan and hemin. Our focus has been on linking these molecules to N<sup>6</sup>-(2-aminoethyl)-FAD to form a conjugate that can be used as a signaling trigger in a displacement assay. A biosensor typically consists of detection and signaling components. The detection aspect of our work involves the use of aptamers, which are single-stranded DNA or RNA molecules that can bind to pre-selected targets, for target recognition. A continuation of our work has been on developing the displacement assay that will utilize the conjugates as a signaling trigger. The displacement assay involves the inactive form of glucose oxidase (Apo-GOx) being reactivated with its cofactor (FAD), which is conjugated to tryptophan or hemin. After GOx is reactivated, the enzymatic activity was measured to determine a dose dependent signal. The N<sup>6</sup>-(2-aminoethyl)-FAD and tryptophan or hemin conjugates were purified via HPLC and characterized using MALDI-TOF. Additional experiments were performed to test the feasibility of detecting the enzyme triggers electrochemically. Future work involves the exploration of the affinity of these conjugates to aptamers, and the data collected will provide a suitable system to explore target binding events leading to co-factor/conjugate release.

Detection of small molecules using redox active enzyme triggers

251st National Meeting (2016)

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