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A novel method for the detection of nitrate was developed using simplified nitrate reductase (SNaR) that was produced by genetic recombination techniques. The SNaR consists of the fragments of the Mo-molybdopterin (MO-MPT) binding site and nitrate reduction active site and has high activity for nitrate reduction. The method is based on a unique combination of the enzyme-catalyzed reduction of nitrate to nitrite by thin-layer coulometry followed by spectroscopic measurement of the colored product generated from the reaction of nitrite with Griess reagents. Coulometric reduction of nitrate to nitrite used methyl viologen (MV(2+)) as the electron transfer mediator for SNaR and controlled potential coulometry in an indium tin oxide (ITO) thin-layer electrochemical cell. Absorbance at 540 nm was proportional to the concentration of nitrate in the sample with a linear range of 1-160 μM and a sensitivity of 8000 AU M(-1). The method required less than 60 μL of sample. Detection of nitrate could also be performed by measuring the charge associated with coulometry. However, the spectroscopic procedure gave superior performance because of interference from the large background charge associated with coulometry. Results for the determination of nitrate concentration in several natural water samples using this device with spectroscopic detection are in good agreement with analysis done with a standard method.

Insulin is stored in pancreatic islets as a zinc-insulin complex, and stimulating the islets results in the release of insulin and zinc. Simulant pancreatic islet beads have been developed using agarose beads (50-250 micro m diameter) derivatized with iminodiacetic acid that have been loaded with zinc. A qualitative comparison of the simulant beads with pancreatic islets has been made by staining with dithizone and a zinc-binding fluorescent dye, TSQ. The binding capacity of simulant beads was determined to be 34 micro mol Zn(2+)/g of dried beads using anodic stripping voltammetry. Hydrochloric acid was used to release zinc from beads to mimic the secretion of insulin from pancreatic islets and a release profile was established. The simulant beads can be used to optimize the islet isolation process and reduce the use of real islets in method development.

Developing sensors for pathogens using real microorganisms for initial testing of the device can introduce additional variables and potential experimental hazards. The artificial microorganism “Bugbead” could be used as a simulant to evaluate the device and for methods development. Bugbead consists of a microsphere coated with proteins to represent the epitopes on a real microorganism. The advantages of the Bugbead include those of being non-pathogenic, similar in bead size to a real microorganism, and having a controllable epitope density. A well-characterized Bugbead eases the study of the device and can be prepared, stored and used safely. Bead-based sandwich immunoassay was done using, as a model system, biotinylated sheep–anti-mouse IgG as a primary antibody immobilized on streptavidin coated 2.8 μm magnetic beads. Neutravidin-dendrimer coated 0.39 μm nonmagnetic Bugbeads were labeled with biotinylated mouse and guinea pig IgGs. Alkaline phosphatase conjugated donkey–anti-guinea pig IgG was the secondary antibody. p -Aminophenyl phosphate was the phosphatase substrate and the p -aminophenol product was detected by rotating disk electrode-amperometry. The minimum detected amount was 360 Bugbeads. The capturing event between magnetic capture beads and Bugbeads was observed by optical microscopy. In simple tests of the concept, an extract containing serotype antigens of E. coli O157:H7 were coupled to Bugbeads and examined in two modified commercial test kits for this pathogen. In both cases positive results was obtained, indicating that the pathogen antigens had been successfully transferred to the microbead and were capable of simulating the assay response to the pathogen itself. These experimental results demonstrate that the Bugbead could be used in the initial steps of developing microbial sensors.

The fluorescence behaviour of human orosomucoid was investigated. The intrinsic fluorescence was more accessible to acrylamide than to the slightly larger succinimide, indicating limited accessibility to part of the tryptophan population. Although I- showed almost no quenching, that of Cs+ was enhanced, and suggested a region of negative charge proximal to an emitting tryptophan residue. Removal of more than 90% of sialic acid from the glycan chains led to no change in the Cs+, I-, succinimide or acrylamide quenching, indicating that the negatively charged region originates with the protein core. Quenching as a function of pH and temperature supported this view. The binding of chlorpromazine monitored by fluorescence quenching, in the presence and in the absence of the small quenching probes (above), led to a model of its binding domain on orosomucoid that includes two tryptophan residues relatively shielded from the bulk solvent, with the third tryptophan residue being on the periphery of the domain, or affected allotopically and near the negatively charged field.