The work presented in this dissertation is the development of a chemical and a biological sensor to detect analytes in water. First, the feasibility of spectroelectrochemical sensing for detecting a metal in different oxidation states is presented. The speciation sensor concept is illustrated with iron in the ferrous (Fe2+) and ferric (Fe3+) oxidation states as the model metal ion. The sensor consists of an indium tin oxide optically transparent electrode (ITO OTE). The ITO side of the electrode is coated with a thin film of cation exchange polymer, Nafion and is loaded with the ligand 2,2’-bipyridine (bipy). Fe2+ in the sample partitions into the film and forms Fe(bipy)32+. The optical response at 520 nm associated with the electrochemical modulation of the Fe(bipy)32+/3+ complex in the film is measured by attenuated total reflectance spectroscopy. The corresponding change in absorbance (ΔA) is proportional to the concentration of Fe2+ in the film, which in turn is proportional to the bulk concentration of Fe2+ in the sample. Fe3+ has more complex coordination chemistry than Fe2+ and is detected indirectly. Fe3+-bipy complexes formed in the film are reduced to Fe(bipy)32+. The corresponding ΔA at 520 nm is proportional to the Fe3+ in the film which in turn is proportional to the concentration of Fe3+ in the sample. Careful manipulation of the applied potential enables speciation of a mixture of Fe2+ and Fe3+. Optimizing film thickness and ligand concentration with respect to the sensor response yields a detection limit of 0.6 × 10-6 M for Fe2+ and 2 × 10-6 M for Fe3+ and a sensor response time of 6 minutes under kinetic measurements.
The second part of the work presents the development of a bead-based enzyme immunoassay to detect the bacterial spores, Bacillus anthracis Sterne strain (Ba). The immunoassay was built by linking biotinylated goat anti Ba antibody to streptavidin coated magnetic beads. Next, γ-irradiated Ba spores were captured by the Ab-coated capture beads. Then β-D-galactosidase conjugated goat anti Ba antibody was attached to the spores to complete the immunoassay.
A small volume electrochemical detection method was developed by using a gold rotating disk electrode. As an alternative method to electrochemical detection, a fluorescence method was also developed. Both detection methods use the same enzyme, β-D-galactosidase but different substrates; para-aminophenyl galactopyranoside in electrochemistry, and fluorescein di-β-D-galactopyranoside in fluorescence. The electrochemical method gave a detection limit of 2.3 × 105 cfu/mL and a linear range from 5.0 × 106 – 1.0 × 108 cfu/mL (R2 = 0.9763) while the fluorescence method gave a lower detection limit of 5.0 × 104 cfu/mL and a linear range from 4.7 × 105 – 1.5 × 107 cfu/mL (R2 = 0.9966). The detection limits were calculated by using three times the standard deviation of the non-specific signal. The non-specific binding (NSB) of the secondary antibody on the capture beads was studied to improve the detection limit of the immunoassay. Blocking agents SuperBlock®, BlockAid™ and Surfactant P20 were tested to treat the beads and compared with beads treated with the reaction buffer, PBS-R (blockers BSA and Tween 20). Results showed that using PBS-R in the original immunoassay was a good choice.
The immunoassay for Ba spores was subjected to some of the components found in finished water supplies. The possible interference of the following components on the immunoassay was systematically evaluated: water hardness, pH, fluoride, soluble iron, organic content, phosphate, residual chlorine and chloramines. Results indicated that only high pH has an effect on the immunoassay at shorter incubation times. Twenty finished water samples collected from around the nation were spiked with Ba spores and tested. Results showed no significant loss of assay sensitivity after incubating 3.05 × 107 cfu/mL of spores in finished water samples.