Methods Development for the Fluorescent Determination of Inorganic Phosphate and Phospho-Proteins

The micronutrient orthophosphate loss into the aquatic environment is one of the primary drivers of harmful algal blooms (HABs) which result in the contamination of drinking water and negatively affects recreation and tourism industries. The aim of this study was to develop fluorescence-based Pi sensors with sensitivity in the low micromolar range Various solid supports were surveyed as potential sensor optimizing components. Quinacrine mustard dihydrochloride bound to Sephadex® LH-20 yielded fluorescence detection of inorganic phosphate concentrations between 0.5-1.25 mM. Quercetin-Al (III), shared a direct negative fluorescence relationship with inorganic phosphate concentrations between 0-1 mM, in aqueous solutions of pH 5. Binding studies of quercetin-Zr (IV) led to an extended investigation for the detection of phospho-biomolecules. Phosphorylation of proteins are prevalent post-translational modifications responsible for signal transduction pathways and gene expression. As such, our quercetin-Zr (IV) probe was studied as a simple and cost-effective stain for phosphorylated proteins immobilized on nitrocellulose membrane. Our studies also explored the potential of an aqueous phosphatase assay with the o-aminobenzoyl-phosphoserine as a potential probe. These studies yielded significant fluorescence differences between phosphorylated and nonphosphorylated L-serine amino acid conjugates in the presence of lanthanide metal, europium (III). These findings can be further utilized for the optimization of inorganic phosphate and phospho-protein/peptide assays