Characterization and optimization of a fiber-optic sensor for detection of enzyme and bacteria

A fiber-optic probe for measurement of enzyme activity has recently been developed in our group. This is being used in various applications including for simple, rapid detection of Escherichia coli ( E. coli) and total coliform bacteria in drinking water. The probe works by extracting fluorescent compounds from solution into a polymer bead on the fiber tip. The goal of this study was to optimize the performance of the fiber-optic probe and to propose a kinetic model for the detection of enzyme activity using the fiber-optic sensor. This kinetic model can then be used to select the best substrates and probe parameters for future probe development.; In the first part of this study, a working model for the uptake of analyte into the probe has been proposed. Uptake experiments for different analytes including 1-hydroxypyrene (Ho-pyr), 2-hydroxyanthracene (Ho-ant), 9-hydroxyphenanthrene (Ho-phe), and 1-hydroxynaphthalene (Ho-naph) were performed to test the validity of the model. This model works well in optimizing the parameters for the probe to obtain best performance, i.e. best sensitivity and response time. According to the model, the uptake rate constant is inversely proportional to the radius of the bead. Uptake signal was found to be proportional to the volume of the bead if the radius of the bead is equal to or less than the radius of the fiber. Therefore, a hemisphere bead with the same radius as the radius of the fiber had the best performance considering both response time and sensitivity. Ho-ant and Ho-pyr were found to be good candidates to be tested when designing enzyme substrates for the fiber-optic probe.; In the second part of the study, detection of different polycyclic aromatic hydrocarbons (PAH), hydroxy PAH (HO-PAH) and methyl-PAH compounds using this fiber-optic probe was studied and the data were analyzed using the uptake model. A strong relationship was observed between the ratio of the uptake rate constant to the desorption rate constant, k2/kd, and the film-solution partition coefficients, Kfs. HO-PAH compounds were found to partition faster than PAH and methyl-PAH compounds, while PAH compounds partitioned into the probe faster than methyl-PAH compounds. NMR studies of ant, Ho-ant, and NH2-ant in different solvents did not indicate hydrogen bonding interactions. Therefore, the faster uptake of HO-PAH and NH2-PAH could be due to higher permeability compared to PAH and methyl-PAH.; Finally, a modified model including enzyme reaction kinetics was proposed for the fiber-optic probe. The reaction between beta-glucuronidase enzyme and two substrates pyrene-beta-glucuronide (pyr-glu) and anthracene-beta-D-glucuronide (ant-glu), was monitored to test this approach. The enzyme reaction model fitted the experimental data reasonably well. Both the slope method and enzyme kinetic model method worked well in the measurement of Km at low enzyme concentrations. Rate constants for the reaction of the enzyme with different substrates can be obtained using the kinetic model method but could not be determined using the slope method. Therefore enzyme activity was best determined by the kinetic model method. Pyr-glu was found to be the best substrate to use when detecting enzyme activity and for the detection of E. coli in a lake water sample.