Iron specificity of a biosensor based on fluorescent pyoverdin immobilized in sol-gel glass

Abstract

Two current technologies used in biosensor development are very promising: 1. The sol-gel process of making microporous glass at room temperature, and 2. Using a fluorescent compound that undergoes fluorescence quenching in response to a specific analyte. These technologies have been combined to produce an iron biosensor. To optimize the iron (II or III) specificity of an iron biosensor, pyoverdin (a fluorescent siderophore produced by Pseudomonas spp.) was immobilized in 3 formulations of porous sol-gel glass. The formulations, A, B, and C, varied in the amount of water added, resulting in respective R values (molar ratio of water:silicon) of 5.6, 8.2, and 10.8. Pyoverdin-doped sol-gel pellets were placed in a flow cell in a fluorometer and the fluorescence quenching was measured as pellets were exposed to 0.28 - 0.56 mM iron (II or III). After 10 minutes of exposure to iron, ferrous ion caused a small fluorescence quenching (89 - 97% of the initial fluorescence, over the range of iron tested) while ferric ion caused much greater quenching (65 - 88%). The most specific and linear response was observed for pyoverdin immobilized in sol-gel C. In contrast, a solution of pyoverdin (3.0 μM) exposed to iron (II or III) for 10 minutes showed an increase in fluorescence (101 - 114%) at low ferrous concentrations (0.45 - 2.18 μM) while exposure to all ferric ion concentrations (0.45 - 3.03 μM) caused quenching. In summary, the iron specificity of pyoverdin was improved by immobilizing it in sol-gel glass C.

Figure 1

Flow cell and flow cell system. a) Flow cell. b) Flow cell system (not to scale).

Figure 2

Copper-chelate chromatogram for sample #5 (P. aeruginosa 15692), showing the large pyoverdin peak (fractions #48 - #56).

Figure 3

Fluorescence and absorbance titrations. A) Fluorescence titration of purified pyoverdin (3 replicates). Data points indicate the fluorescence 10 min. after each iron (III) addition. (ex/em = 390/452 nm) B) Absorbance titration of purified pyoverdin (2 replicates). Data points indicate the absorbance 10 min. after each iron (III) addition.

Figure 4

Leaching of iron (II) from ferrous sulfate-doped sol-gel pellets. The iron concentration initially in the pellets was set to 100%. Data points are the means of 3 replications. Error bars indicate ± standard deviation.

Figure 5

Scanning electron micrographs of: A) sol-gel glass A; B) sol-gel glass B; C) sol-gel glass C. The bar in the lower right of each micrograph is 80 nm.

Figure 6

Fluorescence quenching of pyoverdin immobilized in sol-gel B. 1.0 ml of either 20 mM ferric chloride (Fe³⁺) or 20 mM ferrous sulfate (Fe²⁺) was added at the times indicated by the arrows, for a final iron concentration (II or III) of 0.555 mM. (ex/em = 382/480 nm)

Figure 7

Fluorescence quenching of pyoverdin immobilized in: A) sol-gel A, B) sol-gel B, C) sol-gel C. Data points are mean values of fluorescence 10 min. after the addition of iron (II or III). Error bars indicate ± standard deviation. Means labeled with the same letter are not significantly different (α = 0.05). The initial fluorescent intensity (before addition of iron) was set to 100%. (ex/em = 382/480 nm)

Figure 8

Fluorescence of purified pyoverdin solution (3.0 μM) in response to 70 μl of either 89.5 μM ferric chloride (Fe³⁺) or 89.5 μM ferrous sulfate (Fe²⁺). Each iron solution was added at the time indicated by the arrow, for a final iron (II or III) concentration of 3.0 μM. (ex/em = 390/452 nm)

Figure 9

Fluorescence of purified pyoverdin solution (3.0 μM) in response to ferrous and ferric ion. Data points are mean values of fluorescence 10 min. after the addition of iron (II or III). Error bars indicate ± standard deviation. The initial fluorescent intensity (before addition of iron) was set to 100%. (ex/em = 390/452 nm)