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.

When the fluorescent siderophore pyoverdin (produced by Pseudomonas aeruginosa) binds to a metal ion the fluorescence changes. A pyoverdin solution (in 0.1 M acetate buffer, pH 5.0) was placed in the microwells of a 96-well plate and varying concentrations of the metal cations Al(3+), Ca(2+), Cu(2+), Fe(2+), Fe(3+), Mn(2+), Mg(2+), and Zn(2+) were added. The fluorescence of pyoverdin 60 sec after the addition of an equimolar concentration of metal indicated: (1) no change for Ca(2+), Fe(2+), Mn(2+), Mg(2+), and Zn(2+); (2) a small increase (109%) for Al(3+); (3) decreases in fluorescence for Cu(2+) (83%) and for Fe(3+) (66%). The fluorescence of pyoverdin 24 hr after the addition of equimolar metal indicated: 1) very little change for Ca(2+), Mn(2+), Mg(2+), and Zn(2+); 2) a very large (270%) increase in fluorescence due to Al(3+); 3) an increase (113%) due to Cu(2+); 4) large decreases in fluorescence for both Fe(2+) (15%) and Fe(3+) (0%). Thus, for an iron assay using a free solution of pyoverdin, even with a short (60 sec.) reaction time there can be interference due to Cu(2+), and interference due to high levels of Al(3+).

The Image Algebra Ada (IAA) system is the basis for a programming environment that enables the nearly direct use of image algebra by an Ada programmer. The system has two components: a translator, which converts the IAA source into pure Ada, and runtime support packages for the resulting programs. The most important data structures in this system are images. An image is a map from a set of points (the domain of the image) to values in some Ada type. A point is an n-tuple of integers (any number of dimensions is supported). Points are usually interpreted as being represented in Cartesian coordinates; however, in principle, other coordinate systems, e.g., polar, could be used.A design goal of IAA was to allow arbitrary domains while still supporting "boxy" domains (parailelepipeds) efficiently. A naive strategy for this is to have one representation for boxes, which records the bounds of the box, and one for non-boxes, which is a linear list of the points. The approach taken, however, avoids having two different representations. We decompose the domain into slices along one dimension and use an interval representation for consecutive identical slices. This can represent arbitrary sets and achieves its least space and time costs for boxy sets. The representation is recursive: boxes resemble lists, and nonboxes resemble trees. With this representation, non-boxy domains are fairly compact when they represent areas or volumes rather than scattered points: roughly speaking, the space taken to represent a region is proportional to its boundary size rather than to the size of the point set. We discuss the benefits and costs of such a representation, its impact on the translator implementation, and possible generalizations of the approach.

The leaching behavior of fluorescent siderophore, pyoverdin (iron biosensor), immobilized in acid-catalyzed sol-gel glass is examined. By varying the R value (molar ratio of water:silicon), three different formulations of pyoverdindoped sol-gel glass pellets (R = 5.6, 8.2, 10.8) were made with tetramethyl orthosilicate (TMOS). At three different aging times (2, 4, and 6 weeks), 7-day leaching experiments were conducted on whole and ground pellets. Two different leaching solutions were used: 0.1 M acetate buffer, pH 5.0, and 1 M HCl. Pyoverdin immobilization resulted in conformation changes as suggested by the appearance of two emission peaks centered at 440 and 505 nm. As expected, pyoverdin leached more rapidly from the ground glass; 61 % of the pyoverdin leached from the ground glass within the first 20 minutes, while after 3 hours, only 54 % of the pyoverdin had leached from the whole pellets. As the sol-gel glass aged from 2 to 6 weeks, the initial fluorescence of the ground glass decreased by 34 – 46 % for the three sol-gel formulations, and the 7-day cumulative leachate decreased by 13 – 31 %. However, sol-gel with the lowest R value (5.6) retained pyoverdin better that sol-gels at higher R values. The release of pyoverdin was also characterized by the diffusional exponent (n), which ranged from 0.527 to 0.802 for the ground glass, and from 0.276 to 0.415 for the whole pellets, confirming that whole pellets retained pyoverdin better than ground glass. Taken together, the results suggest for a given sensor configuration, retention of pyoverdin can be optimized with respect to pellet size, R value and aging time.

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