The overall goal of this project is to develop a widely adaptable biosensor for the detection of small-molecules and nucleic acids. The current work focuses on the conjugation of iron protoporphyrin (hemin) to N6-(2-aminoethyl)-FAD (flavin adenine dinucleotide) in order to develop a sensor for hemin. The design for this strategy is dependent on using an aptamer selective for hemin to bind a suboptimal target consisting of a heme-FAD conjugate. Via a displacement reaction the presence of heme will result in an unbound heme-FAD conjugate that can be detected indirectly via a flavoenzyme reactivation or directly via cyclic voltammetry. The displaced conjugate can be used to quantitate the level of heme present in a sample. FAD was selected to serve as the signaling agent for this detection strategy since it is redox active and is an enzyme co-factor necessary for the reactivation of an apo-flavoenzyme. Towards this end N6-(2-aminoethyl)-FAD was prepared by reaction between FAD and aziridine. Subsequently the conjugation of hemin to N6-(2-aminoethyl)-FAD was optimized via changes in reaction conditions to increase purity. The N6-(2-aminoethyl)-FAD precursor and the heme-FAD conjugate reaction crudes were purified via HPLC and characterized via MALDI-Tof. The heme-FAD conjugate was used within enzyme assays to validate the signaling system for the displacement strategy. Additional work exploring the affinity of the heme-FAD conjugates to the aptamer was carried out. These preliminary data provide a suitable system to explore target binding events leading to co-factor release.

Preparation of FAD conjugates to signal target binding events

249th National Meeting (2015)

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