Iron biosensor based on fluorescent pyoverdin
Yoder, Michael Frederic
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Pyoverdin, the fluorescent siderophore produced by Pseudomonas aeruginosagrowing in low iron environments, can be used as an iron biosensor because the3+fluorescence is quenched when it binds to Fe. Other research indicates that pyoverdin2+can also bind to Fe and to other metal ions. With the goal of developing an ironbiosensor using pyoverdin, the research focused on three areas. The first was to compare3+2+the iron specificity (Fe vs. Fe) of pyoverdin in free solution, with pyoverdin that wasimmobilized in three formulations of sol-gel glass. These formulations differed in the Rvalue (molar ratio of water:silicon). The second area was to determine what influencesleaching of pyoverdin from acid-catalyzed sol-gel glass. And, the third area was todetermine the metal ion specificity (for 8 cations commonly found in well water) for afree solution of pyoverdin. The results of these studies showed that for both free and3+2+immobilized pyoverdin there was much greater quenching due to Fe than due to Fe.Immobilizing pyoverdin in sol-gel glass improved the iron specificity, and the sol-gel3+formulation with an R value 10.8 was the most specific for Fe and had the greatestsensitivity. The leaching study showed that as pyoverdin-doped sol-gel glass aged from 2to 4 weeks there was a large reduction in leaching. Differences in leaching due to Rvalue, pH of the leaching solution, and size of the doped sol-gel glass (either ground,sieved pieces or whole pellets) were minor compared to the effects due to aging. Thethird study showed that the fluorescence of a free solution of pyoverdin (0.1 M acetate3+2+buffer, pH 5.0) was rapidly quenched by Fe, but Cu also caused significant3+quenching. When Al was added to pyoverdin there was a small increase in fluorescenceafter 60 sec., but after 24 hours the fluorescence had increased to 270 %. No affect was2+2+2+2+observed for Ca, Mg, Mn, Zn. In summary, the results showed that immobilizingpyoverdin in sol-gel glass can improve the metal ion specificity, but the formulation ofsol-gel glass must be further modified to reduce the amount of leaching.